Evidence for Normal Thyroidal Adenyl Cyclase, Cyclic AMPBinding and Protein-Kinase Activities in Graves' Disease J. ORGIAZZI, I. J. CHOPRA, D. E. WILLIAMS, AND D. H. SOLOMON Department of Medicine, UCLA Center for the Health Sciences, Los Angeles, California ABSTRACT. The adenyl cyclase, cAMP-binding and protein-kinase activities have been studied in thyroid glands from patients with Graves' disease in comparison with normal thyroid glands. The basal and TSH-stimulated adenyl cyclase activities were tested in crude plasma membrane preparations. The characteristics of the intracellular binding of cAMP, i.e., the maximal binding capacity (MBC) for cAMP and affinity constant (Ka) of the binding, and the
basal and cAMP-stimulated protein-kinase activities, were estimated in both the soluble and particulate fractions of thyroid tissue. All of these parameters studied were essentially normal in Graves' disease. It is concluded that hyperthyroidism in Graves' disease is probably not a result of qualitative or quantitative abnormalities in the adenyl cyclase-cAMP protein-kinase system. (/ Clin Endocrinol Metab 40: 248, 1975)
T
HE etiology of Graves disease is still Schwarz-Mann Laboratory (Orangeburg, New not known (1). A causal role of an York),32y-32P-adenosine-5'-triphosphate, sodium abnormal thyroid-stimulator originating salt, ( P-ATP; 1.98 Ci/mmol) from Amershamfrom as yet undefined immunologic ab- Searle (Arlington Heights, Illinois), the nonnormalities and delivered to the thyroid radioactive nucleotides, creatine phosphate and phospho-kinase from Sigma Chemical follicular cells either systemically (LATS, creatine Company (St. Louis, Mo.). The anion exchange LATS-protector, or Human Thyroid Stim- resin Rexyn 202 (C1-SO4), analytical grade ulator) and/or from intrathyroid sources (Fisher Scientific Company, Fair Lawn, New still remains to be established (2-6). It Jersey) was used to separate bound and free has been suggested that abnormalities nucleotides as described by Tsanget al. (8). The in critical thyroid cellular functions, dialysis tubing was seamless regenerated cele.g., adenyl cyclase (AC), intracellular lulose, 24 A average pore size, 1.75-inch diamecAMP binding and/or protein-kinase ter (VWR Scientific, San Diego, Calif). The (PK) activities might be responsible for tubing was used without prior treatment. Calf hyperfunction of the gland in Graves' dis- thymus histone (type II-A) was obtained from Sigma Chemical Co. Toluene containing 10% ease (1,7). In this study, we have evaluated Biosolve Beckman® and 4.2% Liquifluor, New these various activities in thyroid glands of England Nuclear, was used as scintillation fluid. patients with Graves' disease in comparison with normal thyroid glands and have Preparation of tissue fractions found them to be essentially normal. Materials and Methods Materials 3
3
H-Adenosine 3',5'-cyclic monophosphate H-cAMP; 28 Ci/mmol was purchased from
Received September 13, 1974. Supported by grants from USPHS, AM-16155, AM17251, and NIH Career Development Award 1K04 AM-70,225 (Dr. Chopra), and by a fellowship from Ministere des Affaires Etrangeres, France (Dr. Orgiazzi). Address reprints to: Inder J. Chopra, M.D., Department of Medicine, UCLA Center for the Health Sciences, Los Angeles, California, 90024.
Thyroid tissue obtained at surgery or at autopsy within 12 h of death was immediately processed. Thyroid homogenates were prepared in 3 vol (vol/wt) of ice-cold buffer (0.02M Tris, pH 7.5, 0.25M sucrose) using a motor-driven all
glass tissue grinder. Thyroid homogenates were filtered through gauze and 0.5 to 1.0 ml of the filtrate was set aside for estimation of the total RNA and DNA content. The remainder of the filtrates were centrifuged at 105,000 x g for 1 h in a Beckman® L-2 ultracentrifuge at 4 C and cytosols were decanted and stored at - 2 0 C. The pellets were resuspended in tris-sucrose buffer and recentrifuged at 105,000 x g for 1 h. The final pellets were resuspended in 1 vol
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Equilibrium dialysis. Dialysis cells (Technilab) with 5.0 ml capacity on either side of the dialysis membrane were used. 1.5 ml of a solution of thyroid cytosol in buffer (Tris, 6.7 mM,pH 7.7, EDTA, 0.45 mM and theophylline, 5 mM), containing 2.5 mg protein per ml were added to one side of the cell and an equal volume of the same buffer containing a fixed amount of 3H-cAMP ( — 12,000 cpm) and varying Aclenyl cyclase assay quantities of unlabeled cAMP ranging from 0.25 Basal and TSH or NaF-stimulated AC ac- to 200 pmol to the other side. The dialysis cells tivities were determined by a modification of were incubated at 4 C for 24 h. That equilibthe method of Birnbaumer et al. (10); this rium was reached under these conditions was involved measurement of the amount of cAMP shown by the essentially identical amount of generated during incubation of unlabeled ATP radioactivity in the two compartments of the with the AC-containing tissue fraction. Forty /JL\ dialysis cells which contained buffer without of crude thyroid plasma membrane preparation cytosol on either side of dialysis membrane. At containing 0.2-0.5 mg of protein were incu- the end of the incubation, 0.5 ml aliquots were bated at 34 C for 10 min in a final volume of 150 removed from each side of the cell and added to /Ltl; the incubation medium contained Tris 20 vials containing 10 ml of scintillation fluid for mM, pH 7.8, theophylline 10 mM, MgCl2 6mM, radioactivity measurement. The bound cAMP ATP 3 mM, albumin 0.1%, creatine-phosphate was calculated as the difference between the 10 mM and rabbit muscle creatine-kinase 43 fjug. radioactivity in the cytosol side and the radioacThe ratio of MgCl2 to ATP was routinely kept at tivity present in the corresponding buffer side, 2 as suggested by Wolff and Jones (11). Bovine as described by Hiestand et al. (12). No correcTSH (Thytropar®, Armour) dissolved in 0.1% tion for change in volume during dialysis was albumin or sodium fluoride (NaF), dissolved in necessary. Pilot experiments had shown no water were added under a volume of 20 JJL\. change of volume on either side of the dialysis The incubation was terminated by addition cell when the protein concentration of the to the tubes of 200 /xl of cold absolute incubation mixture was 2.5 mg/ml. methanol followed by 2.0 ml of cold absolute ethanol. After incubation for 30 min Competitive protein-binding studies. The pro(or more) in the cold, the tubes were cen- cedure employed was a modification of the trifuged and aliquots of the alcoholic extract cAMP assay described by Tsang et al. (8). delivered into disposable tubes for measureBriefly, five to nine sets of duplicate aliquots of ment of cAMP content by the method described each thyroid cytosol or pellet suspension conbelow using thyroid cytosol as the binder. taining 0.5 to 2.0 mg of protein per ml were Recovery of added cAMP in these conditions equilibrated at 4 C with a fixed amount of was 50 ± 5% (mean ± SEM of 8 experiments). 3 H-cAMP (^6000 cpm) and nonlabeled cAMP Blank tubes were processed in a manner identiranging in amount from 0.25 to 10.0 pmol in cal to that of experimental tubes, except that the buffer (Tris, 6.7 mM, pH 7.7, EDTA, 0.45 mM membrane preparation was added immediately prior to extraction. The quantity of cAMP pres- and theophylline, 5 mM. Final volume of the ent in blank tubes, whenever detectable, was mixture was 1.5 ml. The tubes were incubated subtracted from that measured in the experi- with constant shaking for 20 min, a duration previously determined to be sufficient for mental tubes. equilibrium to be reached. Separation of unbound and bound cAMP was achieved by resin Studies of the binding of cAMP by thyroid adsorption of the unbound nucleotide as retissue ported by Tsang et al. (8). Correction was made Characteristics of cAMP-binding by thyroid for the fraction of the unbound cAMP not tissue were determined by equilibrium dialysis adsorbed by the resin (=5% of total count) by and by competitive protein-binding studies. subtracting from each experimental value the Both techniques gave similar results. The latter, radioactivity remaining after resin treatment of more rapid, was then used routinely. the tubes containing all reagents except that 2.0 (vol/wt of original tissue) of the buffer and stored at —20 C. For AC studies, the mitochondrial fraction from thyroid tissue obtained at surgery was the source of the enzyme; this fraction was prepared as described by Butcher and Serif (9). The mitochondrial fraction has been referred to as crude plasma membrane preparation in this manuscript.
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ORGIAZZI, CHOPRA, WILLIAMS AND SOLOMON
JCE & M • 1975 Vol 40 • No 2
mg/ml of albumin was substituted for thyroid cytosol. In case of equilibrium dialysis, as well as competitive protein binding studies, the bound and free quantities of cAMP were calculated for each total quantity of cAMP in the incubation medium. cAMP endogenously present in cytosol and/or particulate fraction was measured and included in the calculations. Regression line was determined by the least-square method. Maximum binding capacity (MBC) and affinity constant (Ka) were calculated by Scatchard plot analysis (13).
reduce the effect of variations in endogenous cAMP concentrations ( —10~9M) in the assay mixture, exogenous cAMP (10" 9 M) was added to the incubation mixture even for determination of the basal PK activity. In pilot studies, this amount of exogenous cAMP did not influence the basal activity. Finally, PK activity was determined routinely in the presence of 40 fxg of histone, a quantity known from pilot experiments to have no inhibitory effects on the level of enzymatic activity used in this study. For each sample tested, two blank tubes were included to which no histone was added. The radioactivity precipitated in these tubes was subtracted from that in the corresponding exDetermination of protein-kinase (PK) activity perimental tubes. The quantity of phosphorus PK activity was measured in 25 fA of cytosol incorporated into histone was calculated from diluted V4 with water or 25 /JL\ of the 105,000 x g the specific radioactivity of the labeled ATP in pellet which had been suspended in an equal the assay mixture. volume of 50 niM Tris-Cl, pH 7.5, 25 mM KC1 RNA was assayed by the method of Fleck and and 10 mM MgCl2 as described by Garren et al. Begg (19) and DNA by the method of Greenman (14). Protein-phosphokinase activity was as- (20). Endogenous cAMP in thyroid cytosols and sayed as described by Miyamoto et al. (15). In particulate fractions as well as cAMP generated brief, the incubation mixture which had a final during incubation in the AC assay was measvolume of 0.20 ml contained 10 jumol of sodium ured in alcohol extracts by a modification of the glycerol-phosphate buffer, pH 6.5, 0.4 /umol of competitive protein-binding assay (CPBA) of theophylline, 0.06 /Ltmol of ethylene glycerol bis Tsang et al. (8), in which thyroid cytosol was (/3-amino-ethyl ether)-N, N'-tetraacetic acid, 2 substituted for adrenal cytosol as the cAMP /xmol of sodium fluoride, 0.3 nmol of 32P-ATP binder. The modified CPBA for cAMP has (=7 x 105 cpm), 20 nmol of unlabeled ATP, 2 characteristics quite similar to that reported by /amol of magnesium chloride and 40 /xg of calf Tsang et al. Noncyclic nucleotides in amount up thymus histone; cAMP was added to give con- 10"6 mol per tube did not interfere with the centrations ranging from 10"9 to 10"5 M. Incuba- binding of cAMP. None of the thyroid compotion was carried out for 10 min at 34 C in a nents tested (thyroxine, iodine, thyroglobulin) Dubnoff metabolic shaker. The reaction was nor TSH nor NaF had any effect on the CPBA. terminated by addition of 4 ml of 7.5% TCA and Thyroglobulin concentration was measured in 0.2 ml of 0.63% bovine serum albumin and the thyroid cytosols by radioimmunoassay (21) and precipitate obtained by centrifugation was the protein concentration by the method of washed twice by dissolution in I N NaOH and Lowry et al. (22). The difference between the reprecipitation by 5% TCA, as described by two values was taken as the concentration of Yamashita and Field (16). The final precipitate non-thyroglobulin protein in the cytosol. was dissolved in 0.4 ml of 88% formic acid, as suggested by Rapaport and DeGroot (17), and 0.3 Patients ml aliquots added to 10 ml scintillation fluid for determination of radioactivity. Since PK activity Specimens of thyroid glands were obtained was assayed in crude tissue fractions, the final from seven patients aged 18-53 yr undergoing concentration of exogenous ATP was brought to subtotal thyroidectomy for Graves' disease. All 0.1 mM in each tube by adding unlabeled ATP patients were on propylthiouracil and stable in order to reduce the effect on the assay of iodide and were euthyroid at the time of endogenous ATPase activity and of the variation operation. The diagnosis of Graves' disease had in ATP concentration among gland extracts. The been made on the usual clinical and laboratory O.lmM concentration of ATP is at least ten times criteria. Histologic findings were compatible greater than that known to be present in the with Graves' disease in every case. Control thyroid (18). For the same reason, in order to thyroid tissue was obtained surgically from the
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CYCLIC AMP SYSTEM IN GRAVES' DISEASE normal tissue surrounding solitary thyroid nodules in 9 euthyroid patients, aged 17-66 yr. In addition, normal thyroid tissue was obtained at autopsy performed within 12 h after death from 7 patients aged 31-57 yr. In none of these cases did histologic examination show any significant abnormality of the thyroid.
1.4- L 1.2-
0.80.6-
• NORMAL THYROID > GRAVES' DISEASE THYROID
001
01 10 100 BOVINE TSH IN INCUBATION MEDIUM (mU/ml)
100
FIG. 1. Comparison of the effect of TSH on AC activity in crude plasma membrane fraction prepared from Graves' disease and normal thyroid glands.
\
A CPBA \
Ko = O.52x 109 M~1
V
Results AC activity was measured in thyroid tissue removed surgically from two patients with Graves' disease and three with normal tissue (around an adenoma). Baseline AC activity was 43 and 54 pmol of cAMP generated/10 min incubation/mg of protein in the Graves' specimens and 24, 35, and 51 in the normal tissue samples. Although the numbers are too small for valid statistical analysis, there was no striking difference. Similarly, the response of thyroidal AC activity to TSH in Graves' disease was also comparable to that in normal thyroid glands (Fig. 1). In every instance, concentrations of TSH of 0.1 or 0.5 mU/ml significantly stimulated the formation of cAMP. The response of AC activity to 10~2 M NaF was also similar in the Graves' and normal specimens. The ratio of stimulation elicited by 50 mU/ml of TSH and by 10"2 M NaF was 0.22 and 0.74 in the two Graves' thyroids and 0.12, 0.37, and 0.53 in the three normals.
° Equilibriumdialysis
1.0-
f
251
\ V \
109M-
\
Ka = 0.54x
0.40.2-
1.0
2.0
3.0
4.0
5.0
9
B(10" M) FIG. 2. Comparison of the characteristics of the binding of cAMP by thyroid cytosol as determined by equilibrium dialysis and competitive protein binding techniques.
Characteristics of the binding of cAMP were determined in both the cytosol and particulate fractions of thyroid homogenates. When a constant amount of cytosol obtained from the thyroid was incubated in the presence of varying concentrations of cAMP in equilibrium dialysis experiments, the data, plotted and expressed according to Scatchard, showed the existence of a highaffinity low-capacity type of binding site with Ka approximating 1 x 109 M"1 at 4 C and pH 7.7 (Fig. 2). The curve suggested the existence of a second type of binding site(s) of lesser affinity but this was not studied further. Figure 2 also shows a comparison of the estimate of MBC and Ka obtained for the same cytosol by equilibrium dialysis and by competitive proteinbinding methods. The results were comparable. In a study of another thyroid cytosol, Ka values were found to be 0.8 and 0.9 x 109 M"1 and MBC values 33.0 and 29.3 pmol per ml of cytosol by competitive protein-binding and equilibrium dialysis, respectively. Therefore, the more con-
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ORGIAZZI, CHOPRA, WILLIAMS AND SOLOMON
JCE & M . 1975
TABLE 1. Characteristics of the binding of cAMP by thyroid fractions obtained from patients with Graves' disease Condition
Tissue fraction 105,000 x g pellet
Cytosol MBC (pmol of cAMP bound)
MBC (pmol of cAMP bound) Ka
Ka (X1O9M-')
disease
per mg DNA
per mg RNA
(xlO9M-')
per mg DNA
per mg RNA
1 2 3
40.3 23.1 19.2 44.4 22.4 29.9 ± 5.2*
75.0 34.3 50.0 107.3 30.2 59.4 ± 14.3
0.5 0.9 3.3 2.4 2.5
7.8 6.9 1.9
14.4 10.1 3.9
. 1.9 ± 0.5
2.0 4.6 ± 1.5
2.7 7.8 ± 2.7
35.2 ± 5.7
48.8 ± 6.0
2.0 ± 0.3
(3)
(6)
3.8 ± 0.7 (3)
6.3 ± 1.0 (3)
3.7 ± 0.3
(6)
23.3 ± 5.6
35.5 ± 5.4 (5)
1.5 ± 0.4 (7)
2.5 ± 0.5 (4)
5.6 ± 1.2 (4)
2.0 ± 0.4
4
5 Normal Group A (surgical) Group B (autopsy)
(7)
1.1 1.5 3.4 — 4.1
2.5 ± 0.7
(3)
(4)
* Mean ± SE and number of determinations ( ). The mean endogenous cAMP content of cytosol and pellet was respectively 9.4 ± 1.3 pmol/mg DNA and 1.3 ± 0.5 pmol/mg DNA for normal thyroid glands and 6.8 ± 2.3 pmol/mg DNA and 1.0 ± 0.1 pmol/mg DNA for Graves' disease thyroid.
venient competitive protein-binding technique was used for further study of the characteristics of the binding of cAMP. When expressed as pmoles of cAMP bound per gm wet weight of thyroid tissue, the mean MBC value in the cytosols from 5 Graves' disease thyroids, 77 ± 11* was not different from that of 93 ± 14 in the cytosols from 6 normal thyroids obtained at surgery (Group A); however, the values in seven glands obtained at autopsy (Group B), 37 ± 4, were lower than both the Graves' group and group A. In order to reduce the effect of colloid-cell ratio on the interpretation of the data, MBC in cytosol was also expressed as pmoles of cAMP bound per mg of non-thyroglobulin protein. The values 2.4 ± 0.8, 2.1 ± 0.7 and 1.5 ± 0.3 for the Graves disease thyroids, and Group A and B of normal thyroids, respectively, were not different from each other. Finally, data obtained for the soluble and particulate * Mean ± SE here and elsewhere unless indicated otherwise.
fractions were expressed per mg of DNA or per mg of RNA. These are described in Table 1. Again, the mean value in Graves' disease thyroids did not differ significantly from that in groups A or B of normal thyroids. The partition of the cAMP-binder between soluble and particulate fractions was also comparable in normal and Graves' disease thyroids. Also listed in Table 1 are the estimates of the Ka for the binding of cAMP by soluble and particulate fractions of normal and Graves' disease thyroids. There was no difference between the two types of tissues. It was of interest to note that the cAMP-binder in the particulate fraction had similar Ka values to those observed for the binder in the cytosol fraction (Table 1). There was also a significant positive correlation (r = 0.73, P < 0.001) between MBC's for cAMP in the particulate and the soluble fractions. Basal PK activity in the crude soluble and particulate fractions of normal and Graves' disease thyroid glands is reported in Table 2. There were no differences. The
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CYCLIC AMP SYSTEM IN GRAVES' DISEASE
253
TABLE 2. Basal and cAMP-stimulated protein-kinase activity in nmol of phosphorus incorporated in histone per 10 min in thyroid fractions obtained from patients with Graves' disease Tissue fraction 105,00 x g pellet
Cytosol disease
10~9M cAMP
1
10.7
2 3
6.7
4 5
4.9 6.8
Normalf
7.0 ± 1.0* 6.0 ± 1.3 (5)
6.0
10- M cAMP
Increase
1O~ M cAMP
10-5M cAMP
Increase
15.6 10.6 9.6 9.0
4.9 3.9 3.6 4.1
0.24 0.19 0.30
0.32 0.43 0.29
0.08 0.24 0.00
—
—
—
12.0 11.4 ± 1.2 10.8 ± 1.9 (5)
5.2 4.3 ± 0.3 4.8 ± 0.7 (5)
0.11 0.21 ± 0.04 0.17 ± 0.02
0.12 0.29 ± 0.06 0.25 ± 0.01 (3)1
0.01 0.08 ± 0.05 0.08 ± 0.01
5
9
(3)
(3)
Activity of the enzyme is expressed per mg of DNA. * Mean ± SE and number of determinations ( ). \ Results obtained with surgical and autopsy specimens have been pooled.
partition of the activity between soluble and particulate fractions was also similar in the two groups. This was the case whether PK activity was calculated per mg of DNA or RNA. PK activity was also assayed in soluble fractions of normal and Graves' disease thyroids in the presence of increasing concentrations (10~9 to 10~ 5 M) of cAMP. As shown in Table 2, maximally stimulated enzyme activity was similar in both groups. The apparent Km of the enzyme for cAMP was 1.3 ± 0.2 x 10~7 in the normal and 1.7 ± 0.6 x 10~7 in the Graves' disease thyroids; these values were not significantly different from each other. Finally, when PK activity was assayed in the soluble fraction obtained from one normal and one Graves' disease thyroid gland in the presence of amounts of ATP ranging from 1.5 X 10~6M to 3 X 10~4M, the apparent Km of the enzyme for ATP was found to be approximately 3 x 10~5M at minimal as well as maximal cAMP concentration and in both glands. Discussion We have found that the AC, cAMPbinding and PK activities are essentially normal in Graves' disease thyroid glands. Since thyroid glands of patients with Graves' disease had been exposed to propyl-
thiouracil and iodide during preoperative treatment, it would be important to consider the effect of either drug on the results. It has been recently reported (23) that iodine deficiency may augment AC sensitivity to TSH in rat thyroid homogenates. However, no evidence for an inhibition of the activity of this enzyme by high iodine intake has been reported. On the other hand, a study of rat thyroid suggests that propylthiouracil treatment of the animal may tend to enhance thyroidal AC activity (24). In another study, the responsiveness of thyroidal AC to TSH in vitro was reduced by 10~4M iodide and was completely restored by addition of methimazole, 10~4M, to the incubation mixture (25). We observed that AC was normal in the thyroids of our patients. If iodide treatment had significantly inhibited AC activity, one would expect to find increased thyroidal AC activity in specimens obtained from Graves' disease patients who had not received iodide treatment. However, when AC activity was studied in thyroids from Graves' disease patients treated with antithyroid drugs and thyroxine instead of iodide prior to operation, basal thyroidal AC activity was found to be lower, not higher, than that of control tissue from nontoxic goiters (26). Addition-
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254
ORGIAZZI, CHOPRA, WILLIAMS AND SOLOMON
ally, just as in our study, the in vitro AC activity response to TSH in another study was observed to be well preserved in Graves' disease (25). These findings suggest that conventional drug treatment prior to subtotal thyroidectomy probably does not have a major effect on in vitro measurements of thyroid AC activity. Drug treatment of hyperthyroidism apparently has little effect on cAMP binding or PK activities as well. Thus, we observed no effect of iodide, in vitro, up to a concentration of 10~2M, on these parameters. Similarly, propylthiouracil has not been found to modify significantly either basal or stimulated PK activity in the rat (17). Our results are similar to those of other in vitro studies (5,26,27,28) which also suggest that intracellular responses to TSH are essentially normal in Graves' disease. These studies have shown that thyroid AC activity, colloid droplet formation and incorporation of phosphorus into phospholipids respond appreciably to TSH in Graves' disease (5,26,27,28.) Studies of cAMP-binding and PK activity in thyroids from patients with Graves' disease have not been reported previously. Our estimate of the affinity of the binding of cAMP to its receptor in both normal and Graves' disease thyroid is comparable to that reported for other tissues (29). We found the apparent Km of PK for cAMP in Graves' disease, as well as in normal thyroids, to be higher than previously reported for porcine or bovine thyroid (16, 17,30,31). The reason for this discrepancy is not clear. Species differences, the phosphodiesterase activity in the crude thyroid extracts assayed by us for PK activity and/or the addition of ATP in high concentration in the incubation mixture may have contributed to it (32). These factors would, however, not be expected to vitiate the comparison between normal and Graves' disease thyroids studied identically. Our data, in summary, suggest that thyroid hyperfunction in Graves' disease is
JCE & M • 1975 Vol 40 • No 2
probably not the result of an intrinsic abnormality of the AC-cAMP-PK system. While the results of this study add no direct support to the hypothesis suggesting a significant role of an abnormal thyroid stimulator in the etiology of hyperthyroidism in Graves' disease (33), they are not incompatible with it. Acknowledgments We wish to thank Dr. S. Perzik (Cedars of Lebanon Hospital) and the members of the Division of Endocrinology at UCLA for providing us with the thyroid specimens. We are grateful to Dr. A. J. Van Herle and Dr. R. Uller for performing thyroglobulin radioimmunoassays. We appreciate the excellent secretarial assistance of Ms. Barbara Gutowicz.
References 1. Solomon, D. H., I. J. Chopra, Mayo Clin Proc 47: 803, 1972. 2. Adams, D. D., H. D. Purves, Univ Otago Med School Proc 34: 11, 1956. 3. , T. H. Kennedy, / Clin Endocrinol Metab 33: 47, 1971. 4. Shishiba, Y., T. Shimizu, S. Yoshimura, and K. Shizume,/ Clin Endocrinol Metab 36: 517, 1973. 5. Onaya, T., M. Kotani, T. Yamada, and Y. Ochi, J Clin Endocrinol Metab 36: 859, 1973. 6. Volpe, R., M. Edmonds, L. Lamki, P. V. Clarke, and V. V. Row, Mayo Clin Proc 47: 824, 1972. 7. Chopra, I. J., D. H. Solomon, D. E. Johnson, and U. Chopra, Metabolism 19: 760, 1970. 8. Tsang, C. P. W., D. C. Lehotay, and B. E. P. Murphy, J Clin Endocrinol Metab 35: 809, 1972. 9. Butcher, F. R., and G. S. Serif, Biochem Biophys Ada 192: 409, 1969. 10. Birnmaumer, L., S. L. Pohl and M. RodbellJ Biol Chem 244: 3468, 1969. 11. Wolff, J., and A. B. Jones, / Biol Chem 246: 3939, 1971. 12. Hiestand, P. C , V. Eppenberger, and R. A. Jungman, Endocrinology 93: 217, 1973. 13. Scatchard, G., Ann NY Acad Sci 51: 660, 1949. 14". Garren, L. D., G. N. Gill, and G. M. Walton, Ann NY Acad Sci 185: 210, 1971. 15. Miyamoto, E., J. F. Kuo, and P. Greengard,7 Biol Chem 244: 6395, 1969. 16. Yamashita, K. and J. B. Field, Metabolism 21:150, 1972.
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CYCLIC AMP SYSTEM IN GRAVES' DISEASE 17. Rapoport, B., and L. J. DeGroot, Endocrinology 91: 1259, 1972. 18. Field, J. B., P. R. Larsen, K. Yamashita, K. Mashiter, and A. Dekker, J Clin Invest 52: 2404, 1973. 19. Fleck, A., and D. Begg, Biochem Biophys Ada 108: 333, 1965. 20. Greenman, D. L., Endocrinology 87: 716, 1970. 21. Van Herle, A. J., R. P. Uller, N. L. Matthews, and J. Brown. J Clin Invest 52: 1320, 1973. 22. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. RandallJ Biol Chem 193: 265, 1951. 23. Rapoport, B., and S. H. Ingbar, Clin Res 22: 347A, 1974. 24. Zakarija, M., J. M. McKenzie, and C. H. Bastomsky, Endocrinology 92: 1349, 1973. 25. Burke, G.J Clin Endocrinol Metab 30: 76, 1970.
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26. Kendall-Taylor, P., Br MedJ 3: 72, 1973. 27. Schneider, P. B., J Clin Endocrinol Metab 38: 148, 1974. 28. Field, J. B., P. R. Larsen, and K. Yamashita, Trans Assoc Am Phys 86: 300, 1973. 29. Langan, T. A., Advances in Cyclic Nucleotide Research 3: 99, 1973. 30. Rappaport, L., J. F. Leterrier, and J. Nunez, Biochimie 53: 721, 1971. 31. Spaulding, S. W., and G. N. Burrow, Endocrinology 91: 1343, 1972. 32. Haddox, M. K., N. E. Newton, D. K. Hartle, and Goldberg, Biochem Biophys Res Commun 47: 653, 1972. 33. Adams, D. D., T. H. Kennedy, and R. D. Stewart, BrMedJ 2: 199, 1974.
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basal and cAMP-stimulated protein-kinase activities, were estimated in both the soluble and particulate fractions of thyroid tissue. All of these parameters studied were essentially normal in Graves' disease. It is concluded that hyperthyroidism in Graves' disease is probably not a result of qualitative or quantitative abnormalities in the adenyl cyclase-cAMP protein-kinase system. (/ Clin Endocrinol Metab 40: 248, 1975)
T
HE etiology of Graves disease is still Schwarz-Mann Laboratory (Orangeburg, New not known (1). A causal role of an York),32y-32P-adenosine-5'-triphosphate, sodium abnormal thyroid-stimulator originating salt, ( P-ATP; 1.98 Ci/mmol) from Amershamfrom as yet undefined immunologic ab- Searle (Arlington Heights, Illinois), the nonnormalities and delivered to the thyroid radioactive nucleotides, creatine phosphate and phospho-kinase from Sigma Chemical follicular cells either systemically (LATS, creatine Company (St. Louis, Mo.). The anion exchange LATS-protector, or Human Thyroid Stim- resin Rexyn 202 (C1-SO4), analytical grade ulator) and/or from intrathyroid sources (Fisher Scientific Company, Fair Lawn, New still remains to be established (2-6). It Jersey) was used to separate bound and free has been suggested that abnormalities nucleotides as described by Tsanget al. (8). The in critical thyroid cellular functions, dialysis tubing was seamless regenerated cele.g., adenyl cyclase (AC), intracellular lulose, 24 A average pore size, 1.75-inch diamecAMP binding and/or protein-kinase ter (VWR Scientific, San Diego, Calif). The (PK) activities might be responsible for tubing was used without prior treatment. Calf hyperfunction of the gland in Graves' dis- thymus histone (type II-A) was obtained from Sigma Chemical Co. Toluene containing 10% ease (1,7). In this study, we have evaluated Biosolve Beckman® and 4.2% Liquifluor, New these various activities in thyroid glands of England Nuclear, was used as scintillation fluid. patients with Graves' disease in comparison with normal thyroid glands and have Preparation of tissue fractions found them to be essentially normal. Materials and Methods Materials 3
3
H-Adenosine 3',5'-cyclic monophosphate H-cAMP; 28 Ci/mmol was purchased from
Received September 13, 1974. Supported by grants from USPHS, AM-16155, AM17251, and NIH Career Development Award 1K04 AM-70,225 (Dr. Chopra), and by a fellowship from Ministere des Affaires Etrangeres, France (Dr. Orgiazzi). Address reprints to: Inder J. Chopra, M.D., Department of Medicine, UCLA Center for the Health Sciences, Los Angeles, California, 90024.
Thyroid tissue obtained at surgery or at autopsy within 12 h of death was immediately processed. Thyroid homogenates were prepared in 3 vol (vol/wt) of ice-cold buffer (0.02M Tris, pH 7.5, 0.25M sucrose) using a motor-driven all
glass tissue grinder. Thyroid homogenates were filtered through gauze and 0.5 to 1.0 ml of the filtrate was set aside for estimation of the total RNA and DNA content. The remainder of the filtrates were centrifuged at 105,000 x g for 1 h in a Beckman® L-2 ultracentrifuge at 4 C and cytosols were decanted and stored at - 2 0 C. The pellets were resuspended in tris-sucrose buffer and recentrifuged at 105,000 x g for 1 h. The final pellets were resuspended in 1 vol
248
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CYCLIC AMP SYSTEM IN GRAVES' DISEASE
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Equilibrium dialysis. Dialysis cells (Technilab) with 5.0 ml capacity on either side of the dialysis membrane were used. 1.5 ml of a solution of thyroid cytosol in buffer (Tris, 6.7 mM,pH 7.7, EDTA, 0.45 mM and theophylline, 5 mM), containing 2.5 mg protein per ml were added to one side of the cell and an equal volume of the same buffer containing a fixed amount of 3H-cAMP ( — 12,000 cpm) and varying Aclenyl cyclase assay quantities of unlabeled cAMP ranging from 0.25 Basal and TSH or NaF-stimulated AC ac- to 200 pmol to the other side. The dialysis cells tivities were determined by a modification of were incubated at 4 C for 24 h. That equilibthe method of Birnbaumer et al. (10); this rium was reached under these conditions was involved measurement of the amount of cAMP shown by the essentially identical amount of generated during incubation of unlabeled ATP radioactivity in the two compartments of the with the AC-containing tissue fraction. Forty /JL\ dialysis cells which contained buffer without of crude thyroid plasma membrane preparation cytosol on either side of dialysis membrane. At containing 0.2-0.5 mg of protein were incu- the end of the incubation, 0.5 ml aliquots were bated at 34 C for 10 min in a final volume of 150 removed from each side of the cell and added to /Ltl; the incubation medium contained Tris 20 vials containing 10 ml of scintillation fluid for mM, pH 7.8, theophylline 10 mM, MgCl2 6mM, radioactivity measurement. The bound cAMP ATP 3 mM, albumin 0.1%, creatine-phosphate was calculated as the difference between the 10 mM and rabbit muscle creatine-kinase 43 fjug. radioactivity in the cytosol side and the radioacThe ratio of MgCl2 to ATP was routinely kept at tivity present in the corresponding buffer side, 2 as suggested by Wolff and Jones (11). Bovine as described by Hiestand et al. (12). No correcTSH (Thytropar®, Armour) dissolved in 0.1% tion for change in volume during dialysis was albumin or sodium fluoride (NaF), dissolved in necessary. Pilot experiments had shown no water were added under a volume of 20 JJL\. change of volume on either side of the dialysis The incubation was terminated by addition cell when the protein concentration of the to the tubes of 200 /xl of cold absolute incubation mixture was 2.5 mg/ml. methanol followed by 2.0 ml of cold absolute ethanol. After incubation for 30 min Competitive protein-binding studies. The pro(or more) in the cold, the tubes were cen- cedure employed was a modification of the trifuged and aliquots of the alcoholic extract cAMP assay described by Tsang et al. (8). delivered into disposable tubes for measureBriefly, five to nine sets of duplicate aliquots of ment of cAMP content by the method described each thyroid cytosol or pellet suspension conbelow using thyroid cytosol as the binder. taining 0.5 to 2.0 mg of protein per ml were Recovery of added cAMP in these conditions equilibrated at 4 C with a fixed amount of was 50 ± 5% (mean ± SEM of 8 experiments). 3 H-cAMP (^6000 cpm) and nonlabeled cAMP Blank tubes were processed in a manner identiranging in amount from 0.25 to 10.0 pmol in cal to that of experimental tubes, except that the buffer (Tris, 6.7 mM, pH 7.7, EDTA, 0.45 mM membrane preparation was added immediately prior to extraction. The quantity of cAMP pres- and theophylline, 5 mM. Final volume of the ent in blank tubes, whenever detectable, was mixture was 1.5 ml. The tubes were incubated subtracted from that measured in the experi- with constant shaking for 20 min, a duration previously determined to be sufficient for mental tubes. equilibrium to be reached. Separation of unbound and bound cAMP was achieved by resin Studies of the binding of cAMP by thyroid adsorption of the unbound nucleotide as retissue ported by Tsang et al. (8). Correction was made Characteristics of cAMP-binding by thyroid for the fraction of the unbound cAMP not tissue were determined by equilibrium dialysis adsorbed by the resin (=5% of total count) by and by competitive protein-binding studies. subtracting from each experimental value the Both techniques gave similar results. The latter, radioactivity remaining after resin treatment of more rapid, was then used routinely. the tubes containing all reagents except that 2.0 (vol/wt of original tissue) of the buffer and stored at —20 C. For AC studies, the mitochondrial fraction from thyroid tissue obtained at surgery was the source of the enzyme; this fraction was prepared as described by Butcher and Serif (9). The mitochondrial fraction has been referred to as crude plasma membrane preparation in this manuscript.
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ORGIAZZI, CHOPRA, WILLIAMS AND SOLOMON
JCE & M • 1975 Vol 40 • No 2
mg/ml of albumin was substituted for thyroid cytosol. In case of equilibrium dialysis, as well as competitive protein binding studies, the bound and free quantities of cAMP were calculated for each total quantity of cAMP in the incubation medium. cAMP endogenously present in cytosol and/or particulate fraction was measured and included in the calculations. Regression line was determined by the least-square method. Maximum binding capacity (MBC) and affinity constant (Ka) were calculated by Scatchard plot analysis (13).
reduce the effect of variations in endogenous cAMP concentrations ( —10~9M) in the assay mixture, exogenous cAMP (10" 9 M) was added to the incubation mixture even for determination of the basal PK activity. In pilot studies, this amount of exogenous cAMP did not influence the basal activity. Finally, PK activity was determined routinely in the presence of 40 fxg of histone, a quantity known from pilot experiments to have no inhibitory effects on the level of enzymatic activity used in this study. For each sample tested, two blank tubes were included to which no histone was added. The radioactivity precipitated in these tubes was subtracted from that in the corresponding exDetermination of protein-kinase (PK) activity perimental tubes. The quantity of phosphorus PK activity was measured in 25 fA of cytosol incorporated into histone was calculated from diluted V4 with water or 25 /JL\ of the 105,000 x g the specific radioactivity of the labeled ATP in pellet which had been suspended in an equal the assay mixture. volume of 50 niM Tris-Cl, pH 7.5, 25 mM KC1 RNA was assayed by the method of Fleck and and 10 mM MgCl2 as described by Garren et al. Begg (19) and DNA by the method of Greenman (14). Protein-phosphokinase activity was as- (20). Endogenous cAMP in thyroid cytosols and sayed as described by Miyamoto et al. (15). In particulate fractions as well as cAMP generated brief, the incubation mixture which had a final during incubation in the AC assay was measvolume of 0.20 ml contained 10 jumol of sodium ured in alcohol extracts by a modification of the glycerol-phosphate buffer, pH 6.5, 0.4 /umol of competitive protein-binding assay (CPBA) of theophylline, 0.06 /Ltmol of ethylene glycerol bis Tsang et al. (8), in which thyroid cytosol was (/3-amino-ethyl ether)-N, N'-tetraacetic acid, 2 substituted for adrenal cytosol as the cAMP /xmol of sodium fluoride, 0.3 nmol of 32P-ATP binder. The modified CPBA for cAMP has (=7 x 105 cpm), 20 nmol of unlabeled ATP, 2 characteristics quite similar to that reported by /amol of magnesium chloride and 40 /xg of calf Tsang et al. Noncyclic nucleotides in amount up thymus histone; cAMP was added to give con- 10"6 mol per tube did not interfere with the centrations ranging from 10"9 to 10"5 M. Incuba- binding of cAMP. None of the thyroid compotion was carried out for 10 min at 34 C in a nents tested (thyroxine, iodine, thyroglobulin) Dubnoff metabolic shaker. The reaction was nor TSH nor NaF had any effect on the CPBA. terminated by addition of 4 ml of 7.5% TCA and Thyroglobulin concentration was measured in 0.2 ml of 0.63% bovine serum albumin and the thyroid cytosols by radioimmunoassay (21) and precipitate obtained by centrifugation was the protein concentration by the method of washed twice by dissolution in I N NaOH and Lowry et al. (22). The difference between the reprecipitation by 5% TCA, as described by two values was taken as the concentration of Yamashita and Field (16). The final precipitate non-thyroglobulin protein in the cytosol. was dissolved in 0.4 ml of 88% formic acid, as suggested by Rapaport and DeGroot (17), and 0.3 Patients ml aliquots added to 10 ml scintillation fluid for determination of radioactivity. Since PK activity Specimens of thyroid glands were obtained was assayed in crude tissue fractions, the final from seven patients aged 18-53 yr undergoing concentration of exogenous ATP was brought to subtotal thyroidectomy for Graves' disease. All 0.1 mM in each tube by adding unlabeled ATP patients were on propylthiouracil and stable in order to reduce the effect on the assay of iodide and were euthyroid at the time of endogenous ATPase activity and of the variation operation. The diagnosis of Graves' disease had in ATP concentration among gland extracts. The been made on the usual clinical and laboratory O.lmM concentration of ATP is at least ten times criteria. Histologic findings were compatible greater than that known to be present in the with Graves' disease in every case. Control thyroid (18). For the same reason, in order to thyroid tissue was obtained surgically from the
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CYCLIC AMP SYSTEM IN GRAVES' DISEASE normal tissue surrounding solitary thyroid nodules in 9 euthyroid patients, aged 17-66 yr. In addition, normal thyroid tissue was obtained at autopsy performed within 12 h after death from 7 patients aged 31-57 yr. In none of these cases did histologic examination show any significant abnormality of the thyroid.
1.4- L 1.2-
0.80.6-
• NORMAL THYROID > GRAVES' DISEASE THYROID
001
01 10 100 BOVINE TSH IN INCUBATION MEDIUM (mU/ml)
100
FIG. 1. Comparison of the effect of TSH on AC activity in crude plasma membrane fraction prepared from Graves' disease and normal thyroid glands.
\
A CPBA \
Ko = O.52x 109 M~1
V
Results AC activity was measured in thyroid tissue removed surgically from two patients with Graves' disease and three with normal tissue (around an adenoma). Baseline AC activity was 43 and 54 pmol of cAMP generated/10 min incubation/mg of protein in the Graves' specimens and 24, 35, and 51 in the normal tissue samples. Although the numbers are too small for valid statistical analysis, there was no striking difference. Similarly, the response of thyroidal AC activity to TSH in Graves' disease was also comparable to that in normal thyroid glands (Fig. 1). In every instance, concentrations of TSH of 0.1 or 0.5 mU/ml significantly stimulated the formation of cAMP. The response of AC activity to 10~2 M NaF was also similar in the Graves' and normal specimens. The ratio of stimulation elicited by 50 mU/ml of TSH and by 10"2 M NaF was 0.22 and 0.74 in the two Graves' thyroids and 0.12, 0.37, and 0.53 in the three normals.
° Equilibriumdialysis
1.0-
f
251
\ V \
109M-
\
Ka = 0.54x
0.40.2-
1.0
2.0
3.0
4.0
5.0
9
B(10" M) FIG. 2. Comparison of the characteristics of the binding of cAMP by thyroid cytosol as determined by equilibrium dialysis and competitive protein binding techniques.
Characteristics of the binding of cAMP were determined in both the cytosol and particulate fractions of thyroid homogenates. When a constant amount of cytosol obtained from the thyroid was incubated in the presence of varying concentrations of cAMP in equilibrium dialysis experiments, the data, plotted and expressed according to Scatchard, showed the existence of a highaffinity low-capacity type of binding site with Ka approximating 1 x 109 M"1 at 4 C and pH 7.7 (Fig. 2). The curve suggested the existence of a second type of binding site(s) of lesser affinity but this was not studied further. Figure 2 also shows a comparison of the estimate of MBC and Ka obtained for the same cytosol by equilibrium dialysis and by competitive proteinbinding methods. The results were comparable. In a study of another thyroid cytosol, Ka values were found to be 0.8 and 0.9 x 109 M"1 and MBC values 33.0 and 29.3 pmol per ml of cytosol by competitive protein-binding and equilibrium dialysis, respectively. Therefore, the more con-
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252
ORGIAZZI, CHOPRA, WILLIAMS AND SOLOMON
JCE & M . 1975
TABLE 1. Characteristics of the binding of cAMP by thyroid fractions obtained from patients with Graves' disease Condition
Tissue fraction 105,000 x g pellet
Cytosol MBC (pmol of cAMP bound)
MBC (pmol of cAMP bound) Ka
Ka (X1O9M-')
disease
per mg DNA
per mg RNA
(xlO9M-')
per mg DNA
per mg RNA
1 2 3
40.3 23.1 19.2 44.4 22.4 29.9 ± 5.2*
75.0 34.3 50.0 107.3 30.2 59.4 ± 14.3
0.5 0.9 3.3 2.4 2.5
7.8 6.9 1.9
14.4 10.1 3.9
. 1.9 ± 0.5
2.0 4.6 ± 1.5
2.7 7.8 ± 2.7
35.2 ± 5.7
48.8 ± 6.0
2.0 ± 0.3
(3)
(6)
3.8 ± 0.7 (3)
6.3 ± 1.0 (3)
3.7 ± 0.3
(6)
23.3 ± 5.6
35.5 ± 5.4 (5)
1.5 ± 0.4 (7)
2.5 ± 0.5 (4)
5.6 ± 1.2 (4)
2.0 ± 0.4
4
5 Normal Group A (surgical) Group B (autopsy)
(7)
1.1 1.5 3.4 — 4.1
2.5 ± 0.7
(3)
(4)
* Mean ± SE and number of determinations ( ). The mean endogenous cAMP content of cytosol and pellet was respectively 9.4 ± 1.3 pmol/mg DNA and 1.3 ± 0.5 pmol/mg DNA for normal thyroid glands and 6.8 ± 2.3 pmol/mg DNA and 1.0 ± 0.1 pmol/mg DNA for Graves' disease thyroid.
venient competitive protein-binding technique was used for further study of the characteristics of the binding of cAMP. When expressed as pmoles of cAMP bound per gm wet weight of thyroid tissue, the mean MBC value in the cytosols from 5 Graves' disease thyroids, 77 ± 11* was not different from that of 93 ± 14 in the cytosols from 6 normal thyroids obtained at surgery (Group A); however, the values in seven glands obtained at autopsy (Group B), 37 ± 4, were lower than both the Graves' group and group A. In order to reduce the effect of colloid-cell ratio on the interpretation of the data, MBC in cytosol was also expressed as pmoles of cAMP bound per mg of non-thyroglobulin protein. The values 2.4 ± 0.8, 2.1 ± 0.7 and 1.5 ± 0.3 for the Graves disease thyroids, and Group A and B of normal thyroids, respectively, were not different from each other. Finally, data obtained for the soluble and particulate * Mean ± SE here and elsewhere unless indicated otherwise.
fractions were expressed per mg of DNA or per mg of RNA. These are described in Table 1. Again, the mean value in Graves' disease thyroids did not differ significantly from that in groups A or B of normal thyroids. The partition of the cAMP-binder between soluble and particulate fractions was also comparable in normal and Graves' disease thyroids. Also listed in Table 1 are the estimates of the Ka for the binding of cAMP by soluble and particulate fractions of normal and Graves' disease thyroids. There was no difference between the two types of tissues. It was of interest to note that the cAMP-binder in the particulate fraction had similar Ka values to those observed for the binder in the cytosol fraction (Table 1). There was also a significant positive correlation (r = 0.73, P < 0.001) between MBC's for cAMP in the particulate and the soluble fractions. Basal PK activity in the crude soluble and particulate fractions of normal and Graves' disease thyroid glands is reported in Table 2. There were no differences. The
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CYCLIC AMP SYSTEM IN GRAVES' DISEASE
253
TABLE 2. Basal and cAMP-stimulated protein-kinase activity in nmol of phosphorus incorporated in histone per 10 min in thyroid fractions obtained from patients with Graves' disease Tissue fraction 105,00 x g pellet
Cytosol disease
10~9M cAMP
1
10.7
2 3
6.7
4 5
4.9 6.8
Normalf
7.0 ± 1.0* 6.0 ± 1.3 (5)
6.0
10- M cAMP
Increase
1O~ M cAMP
10-5M cAMP
Increase
15.6 10.6 9.6 9.0
4.9 3.9 3.6 4.1
0.24 0.19 0.30
0.32 0.43 0.29
0.08 0.24 0.00
—
—
—
12.0 11.4 ± 1.2 10.8 ± 1.9 (5)
5.2 4.3 ± 0.3 4.8 ± 0.7 (5)
0.11 0.21 ± 0.04 0.17 ± 0.02
0.12 0.29 ± 0.06 0.25 ± 0.01 (3)1
0.01 0.08 ± 0.05 0.08 ± 0.01
5
9
(3)
(3)
Activity of the enzyme is expressed per mg of DNA. * Mean ± SE and number of determinations ( ). \ Results obtained with surgical and autopsy specimens have been pooled.
partition of the activity between soluble and particulate fractions was also similar in the two groups. This was the case whether PK activity was calculated per mg of DNA or RNA. PK activity was also assayed in soluble fractions of normal and Graves' disease thyroids in the presence of increasing concentrations (10~9 to 10~ 5 M) of cAMP. As shown in Table 2, maximally stimulated enzyme activity was similar in both groups. The apparent Km of the enzyme for cAMP was 1.3 ± 0.2 x 10~7 in the normal and 1.7 ± 0.6 x 10~7 in the Graves' disease thyroids; these values were not significantly different from each other. Finally, when PK activity was assayed in the soluble fraction obtained from one normal and one Graves' disease thyroid gland in the presence of amounts of ATP ranging from 1.5 X 10~6M to 3 X 10~4M, the apparent Km of the enzyme for ATP was found to be approximately 3 x 10~5M at minimal as well as maximal cAMP concentration and in both glands. Discussion We have found that the AC, cAMPbinding and PK activities are essentially normal in Graves' disease thyroid glands. Since thyroid glands of patients with Graves' disease had been exposed to propyl-
thiouracil and iodide during preoperative treatment, it would be important to consider the effect of either drug on the results. It has been recently reported (23) that iodine deficiency may augment AC sensitivity to TSH in rat thyroid homogenates. However, no evidence for an inhibition of the activity of this enzyme by high iodine intake has been reported. On the other hand, a study of rat thyroid suggests that propylthiouracil treatment of the animal may tend to enhance thyroidal AC activity (24). In another study, the responsiveness of thyroidal AC to TSH in vitro was reduced by 10~4M iodide and was completely restored by addition of methimazole, 10~4M, to the incubation mixture (25). We observed that AC was normal in the thyroids of our patients. If iodide treatment had significantly inhibited AC activity, one would expect to find increased thyroidal AC activity in specimens obtained from Graves' disease patients who had not received iodide treatment. However, when AC activity was studied in thyroids from Graves' disease patients treated with antithyroid drugs and thyroxine instead of iodide prior to operation, basal thyroidal AC activity was found to be lower, not higher, than that of control tissue from nontoxic goiters (26). Addition-
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254
ORGIAZZI, CHOPRA, WILLIAMS AND SOLOMON
ally, just as in our study, the in vitro AC activity response to TSH in another study was observed to be well preserved in Graves' disease (25). These findings suggest that conventional drug treatment prior to subtotal thyroidectomy probably does not have a major effect on in vitro measurements of thyroid AC activity. Drug treatment of hyperthyroidism apparently has little effect on cAMP binding or PK activities as well. Thus, we observed no effect of iodide, in vitro, up to a concentration of 10~2M, on these parameters. Similarly, propylthiouracil has not been found to modify significantly either basal or stimulated PK activity in the rat (17). Our results are similar to those of other in vitro studies (5,26,27,28) which also suggest that intracellular responses to TSH are essentially normal in Graves' disease. These studies have shown that thyroid AC activity, colloid droplet formation and incorporation of phosphorus into phospholipids respond appreciably to TSH in Graves' disease (5,26,27,28.) Studies of cAMP-binding and PK activity in thyroids from patients with Graves' disease have not been reported previously. Our estimate of the affinity of the binding of cAMP to its receptor in both normal and Graves' disease thyroid is comparable to that reported for other tissues (29). We found the apparent Km of PK for cAMP in Graves' disease, as well as in normal thyroids, to be higher than previously reported for porcine or bovine thyroid (16, 17,30,31). The reason for this discrepancy is not clear. Species differences, the phosphodiesterase activity in the crude thyroid extracts assayed by us for PK activity and/or the addition of ATP in high concentration in the incubation mixture may have contributed to it (32). These factors would, however, not be expected to vitiate the comparison between normal and Graves' disease thyroids studied identically. Our data, in summary, suggest that thyroid hyperfunction in Graves' disease is
JCE & M • 1975 Vol 40 • No 2
probably not the result of an intrinsic abnormality of the AC-cAMP-PK system. While the results of this study add no direct support to the hypothesis suggesting a significant role of an abnormal thyroid stimulator in the etiology of hyperthyroidism in Graves' disease (33), they are not incompatible with it. Acknowledgments We wish to thank Dr. S. Perzik (Cedars of Lebanon Hospital) and the members of the Division of Endocrinology at UCLA for providing us with the thyroid specimens. We are grateful to Dr. A. J. Van Herle and Dr. R. Uller for performing thyroglobulin radioimmunoassays. We appreciate the excellent secretarial assistance of Ms. Barbara Gutowicz.
References 1. Solomon, D. H., I. J. Chopra, Mayo Clin Proc 47: 803, 1972. 2. Adams, D. D., H. D. Purves, Univ Otago Med School Proc 34: 11, 1956. 3. , T. H. Kennedy, / Clin Endocrinol Metab 33: 47, 1971. 4. Shishiba, Y., T. Shimizu, S. Yoshimura, and K. Shizume,/ Clin Endocrinol Metab 36: 517, 1973. 5. Onaya, T., M. Kotani, T. Yamada, and Y. Ochi, J Clin Endocrinol Metab 36: 859, 1973. 6. Volpe, R., M. Edmonds, L. Lamki, P. V. Clarke, and V. V. Row, Mayo Clin Proc 47: 824, 1972. 7. Chopra, I. J., D. H. Solomon, D. E. Johnson, and U. Chopra, Metabolism 19: 760, 1970. 8. Tsang, C. P. W., D. C. Lehotay, and B. E. P. Murphy, J Clin Endocrinol Metab 35: 809, 1972. 9. Butcher, F. R., and G. S. Serif, Biochem Biophys Ada 192: 409, 1969. 10. Birnmaumer, L., S. L. Pohl and M. RodbellJ Biol Chem 244: 3468, 1969. 11. Wolff, J., and A. B. Jones, / Biol Chem 246: 3939, 1971. 12. Hiestand, P. C , V. Eppenberger, and R. A. Jungman, Endocrinology 93: 217, 1973. 13. Scatchard, G., Ann NY Acad Sci 51: 660, 1949. 14". Garren, L. D., G. N. Gill, and G. M. Walton, Ann NY Acad Sci 185: 210, 1971. 15. Miyamoto, E., J. F. Kuo, and P. Greengard,7 Biol Chem 244: 6395, 1969. 16. Yamashita, K. and J. B. Field, Metabolism 21:150, 1972.
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CYCLIC AMP SYSTEM IN GRAVES' DISEASE 17. Rapoport, B., and L. J. DeGroot, Endocrinology 91: 1259, 1972. 18. Field, J. B., P. R. Larsen, K. Yamashita, K. Mashiter, and A. Dekker, J Clin Invest 52: 2404, 1973. 19. Fleck, A., and D. Begg, Biochem Biophys Ada 108: 333, 1965. 20. Greenman, D. L., Endocrinology 87: 716, 1970. 21. Van Herle, A. J., R. P. Uller, N. L. Matthews, and J. Brown. J Clin Invest 52: 1320, 1973. 22. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. RandallJ Biol Chem 193: 265, 1951. 23. Rapoport, B., and S. H. Ingbar, Clin Res 22: 347A, 1974. 24. Zakarija, M., J. M. McKenzie, and C. H. Bastomsky, Endocrinology 92: 1349, 1973. 25. Burke, G.J Clin Endocrinol Metab 30: 76, 1970.
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26. Kendall-Taylor, P., Br MedJ 3: 72, 1973. 27. Schneider, P. B., J Clin Endocrinol Metab 38: 148, 1974. 28. Field, J. B., P. R. Larsen, and K. Yamashita, Trans Assoc Am Phys 86: 300, 1973. 29. Langan, T. A., Advances in Cyclic Nucleotide Research 3: 99, 1973. 30. Rappaport, L., J. F. Leterrier, and J. Nunez, Biochimie 53: 721, 1971. 31. Spaulding, S. W., and G. N. Burrow, Endocrinology 91: 1343, 1972. 32. Haddox, M. K., N. E. Newton, D. K. Hartle, and Goldberg, Biochem Biophys Res Commun 47: 653, 1972. 33. Adams, D. D., T. H. Kennedy, and R. D. Stewart, BrMedJ 2: 199, 1974.
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