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Stimulation of thyroid adenyl cyclase activity and cyclic adenosine 3′,5′-monophosphate by long-acting thyroid stimulator
Stimulation of Thyroid Adenyl Cyclase Activity and Cyclic Adenosine 3’,5’-Monophosphate
by Long-acting
Thyroid Stimulator By TOSHIO KANEKO, URIEL ZOR AND Long-acting thyroid stimulator (LATS) increased adenyl cyclase activity in dog thyroid slices when 3H-adenine incorporation into 3H-cyclic AMP was measured 1W-ATP conversion but not when to 1Gcyclic AMP was used. The former method was demonstrated to be a more sensitive indicator of adenyl cyclase activity, and in the presence of lo-? theophylline as little as 0.25 mU TSH significantly increased adenyl cyclase activity. LATS also increased the concentration of cyclic AMP in thyroid slices, consistent with its activation of adenyl cyclase. Although TSH increased cyclic AMP within three minutes of its addition, LATS did not have any effect until a
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JAMES B. FIELD
30-minute incubation. The effect of LATS increased during longer incubations. LATS did not inhibit phosphodiesterase activity. Papain digestion of LATS produced a fragment which also increased 1%l-glucose oxidation by thyroid slices but still had the same delay in onset of action as LATS. A satisfactory dose response relationship could not be established using the fragment obtained by papain digestion. These results are compatible with the hypothesis that LATS like TSH controls thyroid gland function through the adenyl cyclase-cyclic AMP system. (Metabolism 19: No. 6, June, 430-438, 1970)
URRENT
EVIDENCE SUGGESTS that TSH regulates thyroid gland metabolism as a consequence of stimulation of the enzyme adenyl cyclase and generation of cyclic AMP. 1-S Although it has a different time course of action in vivo, long acting thyroid stimulator (LATS) reproduces most of the effects of TSH on thyroid gland function,“-]” and therefore it has been suggested that its effects are also mediated by the adenyl cyclase-cyclic AMP system.“-‘:’ Bastomsky and McKenzie found that stimulation of IX11 uptake and incorporation into iodothyronine by LATS was augmented by administration of amounts of theophylline which by themselves had no effect.“’ However, the effects of theophylline on thyroid gland metabolism are complex1~14~15 and thus such studies do not provide convincing proof that LATS effects on the thyroid are related to stimulation of cyclic AMP concentrations. The present investigations were done to examine more directly the effect of LATS on thyroid adenyl cyclase activity and cylic AMP generation. From the Clinical Research Unit und the Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pu. Supported by USPHS Grant AM 06865 from the Nationul Institutes of Health. TOSHIO KANEKO, M.D.: Research Associate, Department of Medicine, Uni,,ersity of Pittsburgh School of Medicine, Pittsburgh, Pu. URIEL ZOR, PH.D.: Research Aswciate, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. JA~IES B. FIELD, M.D.: Professor of Medic&e, Department of Medicine. U,>iversit_v of Pittsburgh School of Medicine, Pitfsburgh, Pa. 430
METABOLISM,VOL. 19, No. 6 (JUNE). 1970
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MATERIALS AND METHODS Dog thyroid glands were obtained, sliced and incubated for determination of I%-l-glucose oxidation as previously described .1 Adenyl cyclase activity in thyroid homogenates and slices was measured by previously published methods.1 Tissue cyclic AMP was measured by the method of Kaneko and Field. 1s Conversion of sH-adenine to sH-cyclic AMP by thyroid slices was measured as follows. Slices weighing approximately 20 mg. were incubated for 1 hour at 37’ C in 2 ml. Krebs Ringer bicarbonate buffer (pH 7.4) containing 1 mg./ ml. glucose and albumin and 10 BE. sH-adenine. The gas phase was 95 per cent O,-5 per cent CO,. The slices were then rinsed in a large volume of normal saline and incubated for another 30 minutes in 0.5 ml. of a similar buffer which also contained 10-z M theophylline and the appropriate substances to be tested. 3H-adenine was not added to the buffer during the second incubation. After the second incubation the slices were homogenized in 0.5 ml. 0.1 N HCl and the tube placed in a boiling Ha0 bath for 15 minutes. Following addition of 0.1 ml. of 0.5 M Tris-HCl (pH 7.5) the mixture was neutralized with 0.1 ml. of 0.5 N NaOH. Most of the ATP, ADP, adenine and proteins were removed by precipitation with 0.1 ml. 5 per cent ZnSO, and 0.1 ml. 0.3 N Ba(OH),.tr After centrifugation the supernatant was added to a Dowex 5OW-X4 column and eluted with water. The fraction between 3.5 and 7.5 ml. containing cyclic AMP was collected and reprecipitated twice with ZnSO, and Ba(OH),.lr The clear supernatant was added to 15 ml. Triton X-100 and counted in a liquid scintillation spectrometer. Phosphodiesterase was measured as described previously.1 Theophylline was not present during the incubations and 55 &pmoles of cyclic AMP was added at the beginning of the incubation. Bovine TSH (2 unitsimg.) was a generous gift of the Endocrinology Study Section, National Institutes of Health. LATS was kindly supplied by Dr. Joseph Kriss, Stanford University School of Medicine, Palo Alto, California, and Dr. David Solomon, University of California School of Medicine, Los Angeles, California. The former preparation (LATSK) assayed 50 Kriss units/mg. while the latter preparation (LATS-S) contained 5 Kriss units/mg. Papain-digested LATS (P-LATS) was generously provided by Dr. Joseph Kriss and was prepared according to methods previously published.18 14C-8-ATP (25 mc./mmole) and aH-adenine (22 CJmmole) were obtained from Schwartz BioResearch Corporation. 1JC-l-glucose (2.4 mc./mmole) was purchased from AmershamSearle Corporation. Dowex SOW-X4 was a product of Calbiochemical Corporation. Phosphodiesterase was prepared by the method of Cheung.19 Phosphoenol pyruvate and pyruvate kinase were obtained from Sigma Chemical Corporation. RESULTS
The data in Table 1 demonstrate that LATS (0.5 units) did not increase adenyl cyclase activity in either thyroid homogenate or intact slices when the assay depended upon the conversion of 14C-ATP to 14C-cyclic AMP. TSH (200 mu) and NaF (lo-” M) significantly increased adenyl cyclase activity in both preparations. In other experiments as much as two units of LATS still did not augment adenyl cyclase activity. In contrast LATS increased 3H-adenine incorporation into 3H-cyclic AMP in thyroid slices (Table 2). Similar effects were obtained using two different preparations of LATS. An equivalent amount of y-globulin was ineffective. The stimulation by LATS was probably less than that of 1 mu/ml. TSH and increasing the TSH to 10 mu/ml. gave an even greater effect. NaF (lo-* M) did not increase 3H-adenine incorporation into 3H-cyclic AMP even though it markedly stimulated adenyl cyclase activity as measured by r4C-ATP incorporation into 14C-cylic AMP in both thyroid homogenate and slices. We have previously reported that NaF did not increase cyclic AMP concentrations in thyroid slices. ~JO The data in Table 3 demonstrate that 3H-adenine conversion to “H-cylic AMP is .a sensitive indicator of the
KANEKO,
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Table 1.-Effect -~
of LATS, TSH, and y-Globulin on Adenyl Cyclase Activity in Dog Thyroid Slices and Homogenates .._~ ~__ .__~~~~
__-.
W-ATP Conversion Thyroid Slice
Control
55 k
a-globulin 10 fig. LATS 0.5 units TSH 200 mU NaF 10-a M
63 t 64-1 274 c 565 k
to
“C-cyclic AMP cpttt, 10 mg. Thyroid Homogenate
206 183 193 490 1190
14
10 15 56 146
The results of the adenyl cyclase assay using thyroid slices are the average +- SEM of triplicate determinations and duplicate determinations when thyroid homogenate was used. The incubation medium was 0.15 ml. and contained an ATP regenerating system for both the thyroid slice and homogenate assay. A 30-minute incubation period was used for both. A different dog thyroid was used for the slice and homogenate experiments. Table 2.-Effect
a globulin 6 mg./ml.
Control
1336?120
1584k253
of LATS, TSH, and NaF on 3H-Adenine into 3H-Cyclic AMP by Dog Thyroid Slices LATS-S 2 U/ml.
32322432%
~H-C~CI~~$S~;
cpm/Gm.
Incorporation
2 U/ml.
TSH 1 mU/ml.
TSH 10 mU/ml.
413421164*
6545?1176*
17,356?990+
NaF
10-c M ._._..____
1674-r-398
* p < 0.05. i p < 0.01. The results are the average i SEM of triplicate determinations. Thyroid slices were incubated for 1 hour in 2 ml. Krebs-Ringer bicarbonate buffer containing 1 mg./ml. glucose and albumin and 10 pc. sH-adenine. The slices were then rinsed in saline and incubated for 30 minutes in 0.5 ml. of a similar buffer which also contained 10-z M theophylline and the appropriate substance being tested. Two different LATS preparations were used.
Table 3.-Effect of Various Doses of TSH and Theophylline on 3H Adenine Incorporation into 3H Cyclic AMP in Dog Thyroid Slices Th~or$yl&ine
+
Control 1438+249 2068t273
:‘H Cyclic AMPT;~m~~.m. 0.125
0.25
17442485
19175660 4188?691*
0.5
5
16782974 5464?1069*
10.423-c 1629 t
15
___
10,787+273f
~___
* p < 0.05. t p < 0.01. The results are the average cubated for 1 hour in 2 ml. glucose and albumin and 10 incubated for 30 minutes in theophylline where appropriate.
+ SEM of triplicate determinations. Thyroid slices were inof Krebs-Ringer bicarbonate buffer containing 1 mg./ml. PC. 3H adenine. The slices were then rinsed in saline and 0.5 ml. of a similar buffer which also contained 10-z M P values are based on comparison with the control value.
of adenyl cyclase by TSH when theophylline is also present. Under these conditions as little as 0.25 mU TSH significantly increased 3H-adenine incorporation into 3H-cyclic AMP and 5 mU produced a maximum response. Such five-fold increases are mtich greater than those observed by even larger amounts of TSH when 14C-ATP conversion to 14C-cyclic AMP is used as the basis of adenyl cyclase assay. ls3 Using the latter procedure significant stimulation was usually not obtained with less than 1 mU TSH and increasing effects stimulaticm
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m
Control
m
X-Globulin 6Opg/ml
m
LATS
3 U/ml
TSH
Incubation
Time
in Minutes
Fig. l.-Course of LATS stimulation of CAMP in dog thyroid slices. Results are averages of duplicate determinations. Gamma-globulin was equivalent in weight to LATS.
were still obtained with as much as 200 mU TSH. Without theophylline, 0.25 mU and 0.5 mU TSH did not increase 3H-adenine incorporation into 3H-cyclic AMP. LATS also increased cyclic AMP concentrations in dog thyroid slices (Fig. 1) consistent with its ability to stimulate adenyl cyclase activity. Our previous results indicated that measurement of cyclic AMP levels as compared to adenyl cyclase activity is a much more sensitive indication of TSH action since the per cent increment of the former far exceeds that of the 1atter.l TSH increased cyclic AMP during a ten minute incubation while LATS had no effect at this time. The effect of TSH can be observed as soon as three minutes.20 By 30 minutes LATS significantly increased cyclic AMP concentrations while an equivalent weight of y-globulin was inactive. The stimulation by LATS increased somewhat during a 60-minute incubation but then remained stable for six hours. The effect of TSH was approximately the same during the 6-hour incubation as at the end of the lo-minute incubation. Stimulation by 3 U/ml. LATS was definitely less than that induced by 1 mu/ml. TSH. Such relative potency is similar to that obtained measuring 3H-adenine incorporation into 3H-cyclic AMP, glucose oxidation and 32P incorporation into phospholipids.5 The increased cyclic AMP induced by LATS represents stimulation of adenyl cyclase activity rather than inhibition of degradation of cyclic AMP for the following reasons. LATS had no effect on the phosphodiesterase activity of thyroid homogenate when compared to an equal amount of y-globulin (Fig. 2). The conditions of the assay were such that most of the cyclic AMP present was degraded during the 4-minute incubation period since a small amount of cyclic AMP (55 ppmoles) was added and theophylline was omitted from the incubation. These conditions were felt to be most suitable for demonstrating an effect, if one existed, of LATS
KANEKO,
434
m
Y-Glzb.h
m
LATS
ZOR
AND
FIELD
m&q/ml 5
u/ml
_. lncubotan Time in t&n&s
Fig. 2.-Effect of LATS on phosphodiesterase activity in dog thyroid. Results are averages of duplicate determinations. Fifty-five ppmoles of cyclic AMP were added to initiate reaction. Gamma-globulin was equivalent in weight to LATS. Theophylline was not present during incubation. Table 4.Effect
of Different Amounts of Papain-Digested LATS (P-LATS) on Glucose Oxldation in Dog Thyroid Slices WOz
Control
LATS 1 U/ml.
0.25 mg./ml.
52.97329225
78,564*795*
79,169+6841t
Produced cpm/Gm. P-LATS 1.5 rng./rnl. 0.75 mg./ml. 85,840+2679*
92,954*4074*
y-globulin 1.5 mg./ml.
TSH 1 mu/ml. 66,233+2089’
54,452’28;
* p < 0.01. i p < 0.05. The results are the mean c SEM of triplicate determinations. The incubations were for four hours but 1%l-glucose was only present during the last 45 minutes of the incubation. The
substances
to be tested
were
Table 5.-Effect of Duration (P-LATS) Stimulation Incubation time (minutes)
30
60 1.50
present
throughout
the entire
incubation.
of Incubation on LATS and Papain Digested of Glucose Oxidation in Dog Thyroid Slices
Control
11,968?818 24,285t2139 25,755+541
lLCO~ Produced c m/Gm. LA% 0.5 U/ml. TSH 1 mu/ml.
18,215 C 2592 * 35,724?3995 * 46,917&3968 i
10,665?261 19,363 k 565 33,856r 1.563?
LATS
P-LATS 0.5 mg./ml.
12,133 2 1823 26,165-+1374 34,177+ 1069t
* p < 0.05. t p < 0.01. The results are the mean 2 SEM of triplicate determinations. In the 60- and 150-minute incubations, 14C!-l-glucose was present only during the last 45 minutes of incubation. The substances to be tested were present during the entire period of incubation. on phosphodiesterase. Theophylline inhibited LATS still increased cyclic AMP accumulation ments demonstiated that the TSH stimulation increased cyclase activity rather than inhibition
cyclic AMP degradation1 but in the tissue. Previous experiof cyclic AMP also reflects of phosphodiesterase activity.1
THYROID ADENYL
CYCLASE ACTIVITY
435
Fragments of LATS obtained by papain digestion still augment 1311release from the thyroid in vivo but have a shorter latent period than LATSzl The data in Table 4 demonstrate that such fragments also stimulate glucose oxidation. However, a satisfactory dose response relationship was not established since a six-fold increase in the dose did not produce any greater stimulation of glucose oxidation. Previously we were also unable to demonstrate a satisfactory dose response curve using LATS and glucose oxidation.5 Neither LATS nor papaindigested LATS increased glucose oxidation during 30- or 60-minute incubations but did have a significant effect during a 150-minute incubation (Table 5). In this experiment TSH augmented glucose oxidation during all three incubation periods. DISCUSSION
The present results that LATS increased adenyl cyclase activity and cyclic AMP levels of thyroid slices provide direct support for the suggestion that both LATS and TSH both control thyroid gland function by the same mechanism.“-l3 Gilman and Rall had previously reported that LATS did not increase cyclic AMP during lo- to 60-minute incubations either in the presence or absence of theophylline.2 These investigators used beef thyroid slices while our studies were done with dog thyroid slices. When the effects of TSH on cyclic AMP of thyroid slices are compared, equivalent doses of TSH produced greater per cent increments in dog thyroid slices than in bovine .lJ” This greater sensitivity of dog thyroid slices probably accounts for our positive results. The observation that LATS increases cyclic AMP in thyroid slices is supported by the demonstration that LATS also stimulated “H-adenine incorporation into 3H-cyclic AMP in thyroid slices. Although enzyme assays are usually not done with tissue containing intact cells, the results obtained using 3H-adenine appear to be a good indicator of adenyl cyclase activity. It is possible that accumulation of 3H-cyclic AMP reflects inhibition of its degradation or metabolism rather than stimulation of its formation. However, since LATS did not inhibit phosphodiesterase, alteration of this pathway of cyclic AMP metabolism was not responsible for changes in the concentrations of the nucleotide. The failure of LATS to activate adenyl cyclase in either thyroid homogenates or slices measuring r4C-ATP conversion to ‘“C-cyclic AMP almost certainly reflects the decreased sensitivity of this method. The minimum amount of TSH which stimulated l*C-ATP conversion to l*C-cyclic AMP by thyroid homogenates was 1 mU,1,” while the assay utilizing 3H-adenine was sensitive to as little as 0.25 mu. Since previous in vitro studies indicated that LATS produced effects equivalent to those of less than 1 mU TSH, it is not suprising that LATS did not increase adenyl cyclase activity using an assay whose lower limit of sensitivity is 1 mU TSH. In addition to being more sensitive, the assay utilizing 3H-adenine may be more representative of the physiologic changes in adenyl cyclase taking place in the cell. During the initial incubation with 3H-adenine the intracellular ATP pool is labeled and then subsequently converted to 3H-cyclic AMP during the second incubation. Although NaF was a potent stimulator of adenyl cyclase activity in thyroid homogenates and slices using 14C-ATP and an ATP generating system’*” it did not increase the activity of the enzyme using the “H-
436
KANEKO,
ZOR
AND
FIELD
adenine assay. This last observation is consistent with the failure of NaF to augment cyclic AMP in thyroid slices1 and suggests that NaF stimulation of adenyl cyclase activity may be of no physiologic re1evance.l The stimulation of cyclic AMP by LATS was not as rapid as that produced by TSH. Although the in vivo effects of LATS on Ia11 release from the thyroid are delayed in comparison to those of TSH, there are conflicting reports concerning the time course of the effects on other parameters of thyroid metabolism. Field et al. reported that TSH stimulated both 14C-l-glucose oxidation and 3’P incorporation into phospholipids in dog thyroid slices more rapidly than did LATS.5 In contrast Burke observed no difference in the time of onset of effects of TSH and LATS on glucose oxidation and 32P incorporation.‘” When colloid droplet formation and 1311 release were measured, Shishiba et al. found that TSH and LATS produced similar qualitative changes but that LATS had a longer latent period.7 The fact that LATS did not increase cyclic AMP as rapidly as TSH would be consistent with its slower effect on other parameters of thyroid function. Whether one can explain the marked temporal difference between the peak effect of TSH and LATS on Is11 release in vivo on the basis of the only slightly delayed response of cyclic AMP to LATS remains to be established. It may be important that prolonging the time of incubation did not increase the stimulation of cyclic AMP by TSH while it did when LATS was used. Recent studies of Burke indicate that LATS enters the thyroid cell more slowly than TSHS3 and this may account for its more delayed effect. Adenyl cyclase is probably a membrane-bound enzyme but it is not known whether a substance has to penetrate the cell before it can activate the enzyme. Digestion of LATS with papain produces a fragment which still retains biologic activity but exerts its maximal effect in a much shorter time than does LATS.21,24 This change in time course was attributed to the decreased size of the active fragment. In our experiments measuring glucose oxidation, papain-digested LATS appeared to have the same delayed effect as LATS when compared to TSH. The reason why the fragment from papain digestion does not have a more rapid effect than LATS is not apparent. Since metabolism of LATS and papaindigested LATS is not a factor in the in vitro experiments, the difference in the size of the two stimulators may not be as important as in in vivo studies. Dorrington et al. found that the fragment obtained from papain digestion had equivalent activity to that of LATS when in vitro release of lilll from mouse thyroid gland was evaluated. 25 Under these circumstances renal and other clearance factors would not be operative for either thyroid stimulator. The studies of Burke indicated that LATS either is bound to the thyroid membrane or enters the cell more slowly than does TSHZ3 but data are not available relevant to the fragment obtained from papain digestion. The results measuring glucose oxidation suggest that the fragment from papain digestion is also retarded in entering the cell or being bound to the cell membrane. The failure to obtain a satisfactory dose response curve using papain-digested LATS make it impossible to compare its potency to that of either LATS or TSH. The previous studies which have been done with such fragments have not included dose response experiments. 21,24,25For this reason it is not possible to decide whether the lack of an increasing effect with greater amounts of the
THYROID
ADENYL
CYCLASE
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ACTIVITY
fragment are unique to the parameter of glucose oxidation or not. Although adequate dose response relationships have been obtained using LATS in vivo, we were unable to obtain them when glucose oxidation was assayed.5 The explanation for the absence of dose response relationships is not readily apparent. Nonetheless, in vitro effects of LATS and the fragment obtained after papain digestion appear to be less than that produced by 1 mu/ml. TSH. Although these studies clearly indicate that LATS like TSH stimulates adenyl cyclase activity and cyclic AMP concentrations, they do not necessarily prove that the mechanism of action of either thyroid stimulator must involve this cyclic nucleotide. The data also do not provide any further information as to how cyclic AMP might mediate the changes in thyroid metabolism which have been attributed to either LATS or TSH. ACKNOWLEDGMENT We are grateful to Mrs. Gail Bloom for expert technical
assistance.
REFERENCES 1. Zor, U., Kaneko, T., Lowe, I. P., Bloom, G., and Field, J. B.: Effect of thyroid-stimulating hormone and prostaglandins on thyroid adenyl cyclase activation and cyclic adenosine 3’,5’-monophosphate. J. Biol. Chem. 244:5189, 1969. 2. Gilman, A. G., and Rail, T. W.: Factors influencing adenosine 3’,5’-phosphate accumulation in bovine thyroid slices. J. Biol. Chem. 243:5867, 1968. 3. Pastan, I., and Katzen, R.: Activation of adenyl cyclase in thyroid homogenates by thyroid stimulating hormone. Biochem. Biophys. Res. Commun. 29:972, 1967. 4. Scott, T. W., Good, B. F., and Ferguson, K. A.: Comparative effects of longacting thyroid stimulator and pituitary thyrotropin on the intermediate metabolism of thyroid tissue in vitro. Endocrinology 79:949, 1966. 5. Field, J. B., Remer, A., Bloom, G., and Kriss, J. P.: In vitro stimulation by long-acting thyroid stimulator of thyroid glucose oxidation and s2P incorporation into phospholipids. J. Clin. Invest. 47:1553, 1968. 6. McKenzie, J. M.: Further evidence for a thyroid activator in hyperthyroidism. J. Clin. Endocrinol. 20:380, 1960. 7. Shishiba, Y., Solomon, D. H., and Beall, G. N.: Comparison of the early effects of thyrotropin and long-acting thyroid stimulator on thyroidal secretion. Endocrinology 80:957, 1967. 8. Brown, J., and Munro, D. S.: A new in vitro assay for thyroid-stimulating
hormone. J. Physiol. 182:9, 1966. 9. Ochi, Y., and DeGroot, L. J.: Stimulation of RNA and phospholipid formation by long-acting thyroid stimulator and by thyroid stimulating hormone. Biochim. Biophys. Acta 170:198, 1968. 10. Bastomsky, C. H., and McKenzie, J. M.: Interaction of thyrotropin or the long-acting thyroid stimulator with theophylline. Endocrinology 83: 309, 1968. 11. McKenzie, J. M.: Humoral factors in the pathogenesis of Graves’ Disease. Physiol. Rev. 48:252, 1968. 12. Burke, G.: On the competitive interaction of long-acting thyroid stimulator and thyrotropin in vivo. J. Clin. Endocr. 28: 286, 1968. 13. -: The cell membrane: A common site of action of thyrotropin (TSH) and long-acting thyroid stimulator (LATS). Metabolism 18:720, 1969. 14. Gilman, G. A., and Rall, T. W.: The role of adenosine 3’,5’-phosphate in mediating effects of thyroid-stimulating hormone on carbohydrate metabolism of bovine thyroid slices. J. Biol. Chem. 243:5872, 1968. 15. Zor, U., Bloom, G., Lowe, I. P., and Field, J. B.: Effects of theophylline, prostaglandin E, and adrenergic blocking agents on TSH stimulation of thyroid intermediary metabolism. Endocrinology 84: 1082, 1969. 16. Kaneko, T., and Field, J. B.: A method for determination of 3’,5’ cyclic AMP based on ATP formation. I. Lab. Clin. Med. 74:682, 1969.
438 17. Krishna, G., Weiss, B., and Brodie, B. B.: A simple, sensitive method for the assay of adenyl cyclase. .I. Pharmacol. Exp. Ther. 163 : 379, 1968. 18. Kriss, J. P., Pleshakow, V., and Chien, J. R.: Isolation and identification of the long-acting thyroid stimulator and its relation to hyperthyroidism and circumscribed pretibial myxedema. J. Clin. Endocr. 24: 1005, 1964. 19. Cheung, W. Y.: Properties of cyclic 3’.5’-nucleotide phosphodiesterase from rat brain. Biochemistry 6: 1079, 1967. 20. Kaneko, T., Zor, U., and Field, J. B.: Thyroid-stimulating hormone and prostaof cyclic 3’,5’glandin E, stimulation adenosine monophosphate in thyroid slices. Science 163:1062, 1969. 21. Meek, J. C., Jones, A. E., and Lewis, U. J., and VanderLaan, W. P.: Characteriza-
KANEKO, ZOR AND FIELD tion of the long-acting thyroid stimulator of Graves’ Disease. Proc. Nat. Acad. Sci. 52: 342. 1964. 22. Burke, G.: Comparative effects of thyrotropin and long-acting thyroid stimulator on thyroid intermediary metabolism: relationship to pyridine nucleotide levels. Metabolism 18: 132, 1969. 23. -: Comparison of early effects of thyrotropin and long-acting thyroid stimulator on thyroidal phospholipogenesis. Endocrinology 83: 1210. 1968. 24. Dorrington, K. J.. Carneiro, L.. and Munro. D. S.: The proteolysis of immunoglobulin G with long-acting thyroid stimulating activity. Biochem. J. 98:858, 1966. 25. -, and Munro. D. S.: The longacting thyroid stimulator. Clin. Pharmacol. Ther. 7:7X8, 1966.
by Long-acting
Thyroid Stimulator By TOSHIO KANEKO, URIEL ZOR AND Long-acting thyroid stimulator (LATS) increased adenyl cyclase activity in dog thyroid slices when 3H-adenine incorporation into 3H-cyclic AMP was measured 1W-ATP conversion but not when to 1Gcyclic AMP was used. The former method was demonstrated to be a more sensitive indicator of adenyl cyclase activity, and in the presence of lo-? theophylline as little as 0.25 mU TSH significantly increased adenyl cyclase activity. LATS also increased the concentration of cyclic AMP in thyroid slices, consistent with its activation of adenyl cyclase. Although TSH increased cyclic AMP within three minutes of its addition, LATS did not have any effect until a
C
JAMES B. FIELD
30-minute incubation. The effect of LATS increased during longer incubations. LATS did not inhibit phosphodiesterase activity. Papain digestion of LATS produced a fragment which also increased 1%l-glucose oxidation by thyroid slices but still had the same delay in onset of action as LATS. A satisfactory dose response relationship could not be established using the fragment obtained by papain digestion. These results are compatible with the hypothesis that LATS like TSH controls thyroid gland function through the adenyl cyclase-cyclic AMP system. (Metabolism 19: No. 6, June, 430-438, 1970)
URRENT
EVIDENCE SUGGESTS that TSH regulates thyroid gland metabolism as a consequence of stimulation of the enzyme adenyl cyclase and generation of cyclic AMP. 1-S Although it has a different time course of action in vivo, long acting thyroid stimulator (LATS) reproduces most of the effects of TSH on thyroid gland function,“-]” and therefore it has been suggested that its effects are also mediated by the adenyl cyclase-cyclic AMP system.“-‘:’ Bastomsky and McKenzie found that stimulation of IX11 uptake and incorporation into iodothyronine by LATS was augmented by administration of amounts of theophylline which by themselves had no effect.“’ However, the effects of theophylline on thyroid gland metabolism are complex1~14~15 and thus such studies do not provide convincing proof that LATS effects on the thyroid are related to stimulation of cyclic AMP concentrations. The present investigations were done to examine more directly the effect of LATS on thyroid adenyl cyclase activity and cylic AMP generation. From the Clinical Research Unit und the Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pu. Supported by USPHS Grant AM 06865 from the Nationul Institutes of Health. TOSHIO KANEKO, M.D.: Research Associate, Department of Medicine, Uni,,ersity of Pittsburgh School of Medicine, Pittsburgh, Pu. URIEL ZOR, PH.D.: Research Aswciate, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. JA~IES B. FIELD, M.D.: Professor of Medic&e, Department of Medicine. U,>iversit_v of Pittsburgh School of Medicine, Pitfsburgh, Pa. 430
METABOLISM,VOL. 19, No. 6 (JUNE). 1970
THYROID ADENYL CYCLASE ACTIVITY
431
MATERIALS AND METHODS Dog thyroid glands were obtained, sliced and incubated for determination of I%-l-glucose oxidation as previously described .1 Adenyl cyclase activity in thyroid homogenates and slices was measured by previously published methods.1 Tissue cyclic AMP was measured by the method of Kaneko and Field. 1s Conversion of sH-adenine to sH-cyclic AMP by thyroid slices was measured as follows. Slices weighing approximately 20 mg. were incubated for 1 hour at 37’ C in 2 ml. Krebs Ringer bicarbonate buffer (pH 7.4) containing 1 mg./ ml. glucose and albumin and 10 BE. sH-adenine. The gas phase was 95 per cent O,-5 per cent CO,. The slices were then rinsed in a large volume of normal saline and incubated for another 30 minutes in 0.5 ml. of a similar buffer which also contained 10-z M theophylline and the appropriate substances to be tested. 3H-adenine was not added to the buffer during the second incubation. After the second incubation the slices were homogenized in 0.5 ml. 0.1 N HCl and the tube placed in a boiling Ha0 bath for 15 minutes. Following addition of 0.1 ml. of 0.5 M Tris-HCl (pH 7.5) the mixture was neutralized with 0.1 ml. of 0.5 N NaOH. Most of the ATP, ADP, adenine and proteins were removed by precipitation with 0.1 ml. 5 per cent ZnSO, and 0.1 ml. 0.3 N Ba(OH),.tr After centrifugation the supernatant was added to a Dowex 5OW-X4 column and eluted with water. The fraction between 3.5 and 7.5 ml. containing cyclic AMP was collected and reprecipitated twice with ZnSO, and Ba(OH),.lr The clear supernatant was added to 15 ml. Triton X-100 and counted in a liquid scintillation spectrometer. Phosphodiesterase was measured as described previously.1 Theophylline was not present during the incubations and 55 &pmoles of cyclic AMP was added at the beginning of the incubation. Bovine TSH (2 unitsimg.) was a generous gift of the Endocrinology Study Section, National Institutes of Health. LATS was kindly supplied by Dr. Joseph Kriss, Stanford University School of Medicine, Palo Alto, California, and Dr. David Solomon, University of California School of Medicine, Los Angeles, California. The former preparation (LATSK) assayed 50 Kriss units/mg. while the latter preparation (LATS-S) contained 5 Kriss units/mg. Papain-digested LATS (P-LATS) was generously provided by Dr. Joseph Kriss and was prepared according to methods previously published.18 14C-8-ATP (25 mc./mmole) and aH-adenine (22 CJmmole) were obtained from Schwartz BioResearch Corporation. 1JC-l-glucose (2.4 mc./mmole) was purchased from AmershamSearle Corporation. Dowex SOW-X4 was a product of Calbiochemical Corporation. Phosphodiesterase was prepared by the method of Cheung.19 Phosphoenol pyruvate and pyruvate kinase were obtained from Sigma Chemical Corporation. RESULTS
The data in Table 1 demonstrate that LATS (0.5 units) did not increase adenyl cyclase activity in either thyroid homogenate or intact slices when the assay depended upon the conversion of 14C-ATP to 14C-cyclic AMP. TSH (200 mu) and NaF (lo-” M) significantly increased adenyl cyclase activity in both preparations. In other experiments as much as two units of LATS still did not augment adenyl cyclase activity. In contrast LATS increased 3H-adenine incorporation into 3H-cyclic AMP in thyroid slices (Table 2). Similar effects were obtained using two different preparations of LATS. An equivalent amount of y-globulin was ineffective. The stimulation by LATS was probably less than that of 1 mu/ml. TSH and increasing the TSH to 10 mu/ml. gave an even greater effect. NaF (lo-* M) did not increase 3H-adenine incorporation into 3H-cyclic AMP even though it markedly stimulated adenyl cyclase activity as measured by r4C-ATP incorporation into 14C-cylic AMP in both thyroid homogenate and slices. We have previously reported that NaF did not increase cyclic AMP concentrations in thyroid slices. ~JO The data in Table 3 demonstrate that 3H-adenine conversion to “H-cylic AMP is .a sensitive indicator of the
KANEKO,
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ZOR AND
FIELD
Table 1.-Effect -~
of LATS, TSH, and y-Globulin on Adenyl Cyclase Activity in Dog Thyroid Slices and Homogenates .._~ ~__ .__~~~~
__-.
W-ATP Conversion Thyroid Slice
Control
55 k
a-globulin 10 fig. LATS 0.5 units TSH 200 mU NaF 10-a M
63 t 64-1 274 c 565 k
to
“C-cyclic AMP cpttt, 10 mg. Thyroid Homogenate
206 183 193 490 1190
14
10 15 56 146
The results of the adenyl cyclase assay using thyroid slices are the average +- SEM of triplicate determinations and duplicate determinations when thyroid homogenate was used. The incubation medium was 0.15 ml. and contained an ATP regenerating system for both the thyroid slice and homogenate assay. A 30-minute incubation period was used for both. A different dog thyroid was used for the slice and homogenate experiments. Table 2.-Effect
a globulin 6 mg./ml.
Control
1336?120
1584k253
of LATS, TSH, and NaF on 3H-Adenine into 3H-Cyclic AMP by Dog Thyroid Slices LATS-S 2 U/ml.
32322432%
~H-C~CI~~$S~;
cpm/Gm.
Incorporation
2 U/ml.
TSH 1 mU/ml.
TSH 10 mU/ml.
413421164*
6545?1176*
17,356?990+
NaF
10-c M ._._..____
1674-r-398
* p < 0.05. i p < 0.01. The results are the average i SEM of triplicate determinations. Thyroid slices were incubated for 1 hour in 2 ml. Krebs-Ringer bicarbonate buffer containing 1 mg./ml. glucose and albumin and 10 pc. sH-adenine. The slices were then rinsed in saline and incubated for 30 minutes in 0.5 ml. of a similar buffer which also contained 10-z M theophylline and the appropriate substance being tested. Two different LATS preparations were used.
Table 3.-Effect of Various Doses of TSH and Theophylline on 3H Adenine Incorporation into 3H Cyclic AMP in Dog Thyroid Slices Th~or$yl&ine
+
Control 1438+249 2068t273
:‘H Cyclic AMPT;~m~~.m. 0.125
0.25
17442485
19175660 4188?691*
0.5
5
16782974 5464?1069*
10.423-c 1629 t
15
___
10,787+273f
~___
* p < 0.05. t p < 0.01. The results are the average cubated for 1 hour in 2 ml. glucose and albumin and 10 incubated for 30 minutes in theophylline where appropriate.
+ SEM of triplicate determinations. Thyroid slices were inof Krebs-Ringer bicarbonate buffer containing 1 mg./ml. PC. 3H adenine. The slices were then rinsed in saline and 0.5 ml. of a similar buffer which also contained 10-z M P values are based on comparison with the control value.
of adenyl cyclase by TSH when theophylline is also present. Under these conditions as little as 0.25 mU TSH significantly increased 3H-adenine incorporation into 3H-cyclic AMP and 5 mU produced a maximum response. Such five-fold increases are mtich greater than those observed by even larger amounts of TSH when 14C-ATP conversion to 14C-cyclic AMP is used as the basis of adenyl cyclase assay. ls3 Using the latter procedure significant stimulation was usually not obtained with less than 1 mU TSH and increasing effects stimulaticm
THYROID
ADENYL
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ACTIVITY
m
Control
m
X-Globulin 6Opg/ml
m
LATS
3 U/ml
TSH
Incubation
Time
in Minutes
Fig. l.-Course of LATS stimulation of CAMP in dog thyroid slices. Results are averages of duplicate determinations. Gamma-globulin was equivalent in weight to LATS.
were still obtained with as much as 200 mU TSH. Without theophylline, 0.25 mU and 0.5 mU TSH did not increase 3H-adenine incorporation into 3H-cyclic AMP. LATS also increased cyclic AMP concentrations in dog thyroid slices (Fig. 1) consistent with its ability to stimulate adenyl cyclase activity. Our previous results indicated that measurement of cyclic AMP levels as compared to adenyl cyclase activity is a much more sensitive indication of TSH action since the per cent increment of the former far exceeds that of the 1atter.l TSH increased cyclic AMP during a ten minute incubation while LATS had no effect at this time. The effect of TSH can be observed as soon as three minutes.20 By 30 minutes LATS significantly increased cyclic AMP concentrations while an equivalent weight of y-globulin was inactive. The stimulation by LATS increased somewhat during a 60-minute incubation but then remained stable for six hours. The effect of TSH was approximately the same during the 6-hour incubation as at the end of the lo-minute incubation. Stimulation by 3 U/ml. LATS was definitely less than that induced by 1 mu/ml. TSH. Such relative potency is similar to that obtained measuring 3H-adenine incorporation into 3H-cyclic AMP, glucose oxidation and 32P incorporation into phospholipids.5 The increased cyclic AMP induced by LATS represents stimulation of adenyl cyclase activity rather than inhibition of degradation of cyclic AMP for the following reasons. LATS had no effect on the phosphodiesterase activity of thyroid homogenate when compared to an equal amount of y-globulin (Fig. 2). The conditions of the assay were such that most of the cyclic AMP present was degraded during the 4-minute incubation period since a small amount of cyclic AMP (55 ppmoles) was added and theophylline was omitted from the incubation. These conditions were felt to be most suitable for demonstrating an effect, if one existed, of LATS
KANEKO,
434
m
Y-Glzb.h
m
LATS
ZOR
AND
FIELD
m&q/ml 5
u/ml
_. lncubotan Time in t&n&s
Fig. 2.-Effect of LATS on phosphodiesterase activity in dog thyroid. Results are averages of duplicate determinations. Fifty-five ppmoles of cyclic AMP were added to initiate reaction. Gamma-globulin was equivalent in weight to LATS. Theophylline was not present during incubation. Table 4.Effect
of Different Amounts of Papain-Digested LATS (P-LATS) on Glucose Oxldation in Dog Thyroid Slices WOz
Control
LATS 1 U/ml.
0.25 mg./ml.
52.97329225
78,564*795*
79,169+6841t
Produced cpm/Gm. P-LATS 1.5 rng./rnl. 0.75 mg./ml. 85,840+2679*
92,954*4074*
y-globulin 1.5 mg./ml.
TSH 1 mu/ml. 66,233+2089’
54,452’28;
* p < 0.01. i p < 0.05. The results are the mean c SEM of triplicate determinations. The incubations were for four hours but 1%l-glucose was only present during the last 45 minutes of the incubation. The
substances
to be tested
were
Table 5.-Effect of Duration (P-LATS) Stimulation Incubation time (minutes)
30
60 1.50
present
throughout
the entire
incubation.
of Incubation on LATS and Papain Digested of Glucose Oxidation in Dog Thyroid Slices
Control
11,968?818 24,285t2139 25,755+541
lLCO~ Produced c m/Gm. LA% 0.5 U/ml. TSH 1 mu/ml.
18,215 C 2592 * 35,724?3995 * 46,917&3968 i
10,665?261 19,363 k 565 33,856r 1.563?
LATS
P-LATS 0.5 mg./ml.
12,133 2 1823 26,165-+1374 34,177+ 1069t
* p < 0.05. t p < 0.01. The results are the mean 2 SEM of triplicate determinations. In the 60- and 150-minute incubations, 14C!-l-glucose was present only during the last 45 minutes of incubation. The substances to be tested were present during the entire period of incubation. on phosphodiesterase. Theophylline inhibited LATS still increased cyclic AMP accumulation ments demonstiated that the TSH stimulation increased cyclase activity rather than inhibition
cyclic AMP degradation1 but in the tissue. Previous experiof cyclic AMP also reflects of phosphodiesterase activity.1
THYROID ADENYL
CYCLASE ACTIVITY
435
Fragments of LATS obtained by papain digestion still augment 1311release from the thyroid in vivo but have a shorter latent period than LATSzl The data in Table 4 demonstrate that such fragments also stimulate glucose oxidation. However, a satisfactory dose response relationship was not established since a six-fold increase in the dose did not produce any greater stimulation of glucose oxidation. Previously we were also unable to demonstrate a satisfactory dose response curve using LATS and glucose oxidation.5 Neither LATS nor papaindigested LATS increased glucose oxidation during 30- or 60-minute incubations but did have a significant effect during a 150-minute incubation (Table 5). In this experiment TSH augmented glucose oxidation during all three incubation periods. DISCUSSION
The present results that LATS increased adenyl cyclase activity and cyclic AMP levels of thyroid slices provide direct support for the suggestion that both LATS and TSH both control thyroid gland function by the same mechanism.“-l3 Gilman and Rall had previously reported that LATS did not increase cyclic AMP during lo- to 60-minute incubations either in the presence or absence of theophylline.2 These investigators used beef thyroid slices while our studies were done with dog thyroid slices. When the effects of TSH on cyclic AMP of thyroid slices are compared, equivalent doses of TSH produced greater per cent increments in dog thyroid slices than in bovine .lJ” This greater sensitivity of dog thyroid slices probably accounts for our positive results. The observation that LATS increases cyclic AMP in thyroid slices is supported by the demonstration that LATS also stimulated “H-adenine incorporation into 3H-cyclic AMP in thyroid slices. Although enzyme assays are usually not done with tissue containing intact cells, the results obtained using 3H-adenine appear to be a good indicator of adenyl cyclase activity. It is possible that accumulation of 3H-cyclic AMP reflects inhibition of its degradation or metabolism rather than stimulation of its formation. However, since LATS did not inhibit phosphodiesterase, alteration of this pathway of cyclic AMP metabolism was not responsible for changes in the concentrations of the nucleotide. The failure of LATS to activate adenyl cyclase in either thyroid homogenates or slices measuring r4C-ATP conversion to ‘“C-cyclic AMP almost certainly reflects the decreased sensitivity of this method. The minimum amount of TSH which stimulated l*C-ATP conversion to l*C-cyclic AMP by thyroid homogenates was 1 mU,1,” while the assay utilizing 3H-adenine was sensitive to as little as 0.25 mu. Since previous in vitro studies indicated that LATS produced effects equivalent to those of less than 1 mU TSH, it is not suprising that LATS did not increase adenyl cyclase activity using an assay whose lower limit of sensitivity is 1 mU TSH. In addition to being more sensitive, the assay utilizing 3H-adenine may be more representative of the physiologic changes in adenyl cyclase taking place in the cell. During the initial incubation with 3H-adenine the intracellular ATP pool is labeled and then subsequently converted to 3H-cyclic AMP during the second incubation. Although NaF was a potent stimulator of adenyl cyclase activity in thyroid homogenates and slices using 14C-ATP and an ATP generating system’*” it did not increase the activity of the enzyme using the “H-
436
KANEKO,
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AND
FIELD
adenine assay. This last observation is consistent with the failure of NaF to augment cyclic AMP in thyroid slices1 and suggests that NaF stimulation of adenyl cyclase activity may be of no physiologic re1evance.l The stimulation of cyclic AMP by LATS was not as rapid as that produced by TSH. Although the in vivo effects of LATS on Ia11 release from the thyroid are delayed in comparison to those of TSH, there are conflicting reports concerning the time course of the effects on other parameters of thyroid metabolism. Field et al. reported that TSH stimulated both 14C-l-glucose oxidation and 3’P incorporation into phospholipids in dog thyroid slices more rapidly than did LATS.5 In contrast Burke observed no difference in the time of onset of effects of TSH and LATS on glucose oxidation and 32P incorporation.‘” When colloid droplet formation and 1311 release were measured, Shishiba et al. found that TSH and LATS produced similar qualitative changes but that LATS had a longer latent period.7 The fact that LATS did not increase cyclic AMP as rapidly as TSH would be consistent with its slower effect on other parameters of thyroid function. Whether one can explain the marked temporal difference between the peak effect of TSH and LATS on Is11 release in vivo on the basis of the only slightly delayed response of cyclic AMP to LATS remains to be established. It may be important that prolonging the time of incubation did not increase the stimulation of cyclic AMP by TSH while it did when LATS was used. Recent studies of Burke indicate that LATS enters the thyroid cell more slowly than TSHS3 and this may account for its more delayed effect. Adenyl cyclase is probably a membrane-bound enzyme but it is not known whether a substance has to penetrate the cell before it can activate the enzyme. Digestion of LATS with papain produces a fragment which still retains biologic activity but exerts its maximal effect in a much shorter time than does LATS.21,24 This change in time course was attributed to the decreased size of the active fragment. In our experiments measuring glucose oxidation, papain-digested LATS appeared to have the same delayed effect as LATS when compared to TSH. The reason why the fragment from papain digestion does not have a more rapid effect than LATS is not apparent. Since metabolism of LATS and papaindigested LATS is not a factor in the in vitro experiments, the difference in the size of the two stimulators may not be as important as in in vivo studies. Dorrington et al. found that the fragment obtained from papain digestion had equivalent activity to that of LATS when in vitro release of lilll from mouse thyroid gland was evaluated. 25 Under these circumstances renal and other clearance factors would not be operative for either thyroid stimulator. The studies of Burke indicated that LATS either is bound to the thyroid membrane or enters the cell more slowly than does TSHZ3 but data are not available relevant to the fragment obtained from papain digestion. The results measuring glucose oxidation suggest that the fragment from papain digestion is also retarded in entering the cell or being bound to the cell membrane. The failure to obtain a satisfactory dose response curve using papain-digested LATS make it impossible to compare its potency to that of either LATS or TSH. The previous studies which have been done with such fragments have not included dose response experiments. 21,24,25For this reason it is not possible to decide whether the lack of an increasing effect with greater amounts of the
THYROID
ADENYL
CYCLASE
437
ACTIVITY
fragment are unique to the parameter of glucose oxidation or not. Although adequate dose response relationships have been obtained using LATS in vivo, we were unable to obtain them when glucose oxidation was assayed.5 The explanation for the absence of dose response relationships is not readily apparent. Nonetheless, in vitro effects of LATS and the fragment obtained after papain digestion appear to be less than that produced by 1 mu/ml. TSH. Although these studies clearly indicate that LATS like TSH stimulates adenyl cyclase activity and cyclic AMP concentrations, they do not necessarily prove that the mechanism of action of either thyroid stimulator must involve this cyclic nucleotide. The data also do not provide any further information as to how cyclic AMP might mediate the changes in thyroid metabolism which have been attributed to either LATS or TSH. ACKNOWLEDGMENT We are grateful to Mrs. Gail Bloom for expert technical
assistance.
REFERENCES 1. Zor, U., Kaneko, T., Lowe, I. P., Bloom, G., and Field, J. B.: Effect of thyroid-stimulating hormone and prostaglandins on thyroid adenyl cyclase activation and cyclic adenosine 3’,5’-monophosphate. J. Biol. Chem. 244:5189, 1969. 2. Gilman, A. G., and Rail, T. W.: Factors influencing adenosine 3’,5’-phosphate accumulation in bovine thyroid slices. J. Biol. Chem. 243:5867, 1968. 3. Pastan, I., and Katzen, R.: Activation of adenyl cyclase in thyroid homogenates by thyroid stimulating hormone. Biochem. Biophys. Res. Commun. 29:972, 1967. 4. Scott, T. W., Good, B. F., and Ferguson, K. A.: Comparative effects of longacting thyroid stimulator and pituitary thyrotropin on the intermediate metabolism of thyroid tissue in vitro. Endocrinology 79:949, 1966. 5. Field, J. B., Remer, A., Bloom, G., and Kriss, J. P.: In vitro stimulation by long-acting thyroid stimulator of thyroid glucose oxidation and s2P incorporation into phospholipids. J. Clin. Invest. 47:1553, 1968. 6. McKenzie, J. M.: Further evidence for a thyroid activator in hyperthyroidism. J. Clin. Endocrinol. 20:380, 1960. 7. Shishiba, Y., Solomon, D. H., and Beall, G. N.: Comparison of the early effects of thyrotropin and long-acting thyroid stimulator on thyroidal secretion. Endocrinology 80:957, 1967. 8. Brown, J., and Munro, D. S.: A new in vitro assay for thyroid-stimulating
hormone. J. Physiol. 182:9, 1966. 9. Ochi, Y., and DeGroot, L. J.: Stimulation of RNA and phospholipid formation by long-acting thyroid stimulator and by thyroid stimulating hormone. Biochim. Biophys. Acta 170:198, 1968. 10. Bastomsky, C. H., and McKenzie, J. M.: Interaction of thyrotropin or the long-acting thyroid stimulator with theophylline. Endocrinology 83: 309, 1968. 11. McKenzie, J. M.: Humoral factors in the pathogenesis of Graves’ Disease. Physiol. Rev. 48:252, 1968. 12. Burke, G.: On the competitive interaction of long-acting thyroid stimulator and thyrotropin in vivo. J. Clin. Endocr. 28: 286, 1968. 13. -: The cell membrane: A common site of action of thyrotropin (TSH) and long-acting thyroid stimulator (LATS). Metabolism 18:720, 1969. 14. Gilman, G. A., and Rall, T. W.: The role of adenosine 3’,5’-phosphate in mediating effects of thyroid-stimulating hormone on carbohydrate metabolism of bovine thyroid slices. J. Biol. Chem. 243:5872, 1968. 15. Zor, U., Bloom, G., Lowe, I. P., and Field, J. B.: Effects of theophylline, prostaglandin E, and adrenergic blocking agents on TSH stimulation of thyroid intermediary metabolism. Endocrinology 84: 1082, 1969. 16. Kaneko, T., and Field, J. B.: A method for determination of 3’,5’ cyclic AMP based on ATP formation. I. Lab. Clin. Med. 74:682, 1969.
438 17. Krishna, G., Weiss, B., and Brodie, B. B.: A simple, sensitive method for the assay of adenyl cyclase. .I. Pharmacol. Exp. Ther. 163 : 379, 1968. 18. Kriss, J. P., Pleshakow, V., and Chien, J. R.: Isolation and identification of the long-acting thyroid stimulator and its relation to hyperthyroidism and circumscribed pretibial myxedema. J. Clin. Endocr. 24: 1005, 1964. 19. Cheung, W. Y.: Properties of cyclic 3’.5’-nucleotide phosphodiesterase from rat brain. Biochemistry 6: 1079, 1967. 20. Kaneko, T., Zor, U., and Field, J. B.: Thyroid-stimulating hormone and prostaof cyclic 3’,5’glandin E, stimulation adenosine monophosphate in thyroid slices. Science 163:1062, 1969. 21. Meek, J. C., Jones, A. E., and Lewis, U. J., and VanderLaan, W. P.: Characteriza-
KANEKO, ZOR AND FIELD tion of the long-acting thyroid stimulator of Graves’ Disease. Proc. Nat. Acad. Sci. 52: 342. 1964. 22. Burke, G.: Comparative effects of thyrotropin and long-acting thyroid stimulator on thyroid intermediary metabolism: relationship to pyridine nucleotide levels. Metabolism 18: 132, 1969. 23. -: Comparison of early effects of thyrotropin and long-acting thyroid stimulator on thyroidal phospholipogenesis. Endocrinology 83: 1210. 1968. 24. Dorrington, K. J.. Carneiro, L.. and Munro. D. S.: The proteolysis of immunoglobulin G with long-acting thyroid stimulating activity. Biochem. J. 98:858, 1966. 25. -, and Munro. D. S.: The longacting thyroid stimulator. Clin. Pharmacol. Ther. 7:7X8, 1966.