FirstDepartment of Internal Medicine, School of Medicine, Gunma University, Maebashi. Endocrinol. Japon. 1960, 7(3),

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1 Endocrinol. Japon. 1960, 7(3), COINCIDENTAL REBOUND PHENOMENA OF THYROID FUNCTION AND NEUROSECRETORY SUBSTANCE IN THE NEUROHYPOPHYSIS FOLLOWING METHYL- THIOURACIL OR THYROXINE WITHDRAWAL IN THE RATS SHIN-ICHI SHIMODA FirstDepartment of Internal Medicine, School of Medicine, Gunma University, Maebashi There are many bits of evidence that the pituitary-thyroid axisaffords an example of a physiological servo mechanism(hoskins, 1949). When the titer of circulating thyroxine rises, the anterior pituitary is selectively inhibited and the discharge of thyrotropin is thereby decreased. Contrariwise, episodic or persistent thyroxine deficiency results in augmented thyrotropin production with resulting tendency for the production of more thyroid hormone(d'angelo, 1954; Brown- Grant, 1957). This reciprocal relationship has been considered as a major factor for the production of rebound phenomena of thyroid function which appears after the removal of thyroid depressant such as antithyroid drugs or thyroxine. However, relatively little is yet known as to whether the rebound phenomena produced by antithyroid drugs or by thyroxine are essentially similar in nature. Although the reciprocal relationship between the thyroid and the pituitary gland "thermostaticcontrol"is is of great importance, not it adequate has become to account increasingly for many evident phenomena that this type which of have been observed(bogdanove and Halmi, 1953; Harris, 1955; Ganong et al., 1955; Goldberg et al., 1956; Greer, 1957; Harris and Woods, 1958; Yamada, 1959). The evidence suggests that the hypothalamus controls pituitary thyrotropin secretion through some unknown neurohumoral material(s)which passes through the hypophyseal portal vessels from the median eminence of the hypothalamus to the anterior pituitary(harris, 1955, 1956; Hild, 1956), but the exact nature of this hypothalamic control is not known at present. There is the considerable body of evidence that neurosecretory material of the hypothalamo-hypophyseal system is concerned in the control of pituitary thyrotropin secretion(herring, 1908; Shibusawa et al., 1956; Shiozaki, 1956; Yamada, 1957; Azzali and Shernin, 1958; Shimizu, 1959; Shichijo et al., 1959). Because of these uncertainties it seemes of interest to compare the effect of methylthiouracil(mtu)withdrawal on the pituitary-thyroid axis with that of thyroxine and to study the possible participation of neurosecretory system in the hypothalamus for the production of rebound phenomena in the pituitary-thyroid axis. Received for publication April 30, 1960,

2 SHIMODA Vol.7, No.3 MATERIALS AND METHODS One hundred and fifty nine adult male rats of Wistar strain weighing 100 `470g were divided into 2 groups, one treated with methylthiouracil(mtu)and the other with L-thyroxine (T4). MTU group Forty two animals were injected with 40mg of MTU daily intraperitoneally for 5 days. Twenty five animals served as control. 131I(20ƒÊC)was injected intraperitoneally 24 hrs. before the autopsy. They were killed by decapitation at 1, 2, 3, 4, 7 and 11 days after the last MTU treatment. Blood was collected to measure the conversion ratio by the method of Dougherty et al. (1951). Immediately after sacrifice, the pituitary was removed, fixed with Zenkel-formol solution, cut in 4ƒÊ thickness in sagittal planes after routine paraffin technique and stained by Gomori's chrome-alum hematoxylin and phloxine method. Thyroid gland was dissected cleanly and weighed on a torsion balance. Thyroid weight per 100g body weight was culculated from the final body weight and expressed as per cent of the control. The radioactivity in the thyroid was measured with a scintillation counter and the 131I uptake was expressed as per cent of the control. Thirty animals received only single injection of MTU(40mg)intraperitoneally, and 15 animals served as the control. They were killed at 6, 12, 24 and 48hrs., 7 and 11 days after the injection. 131I was injected 6 and 12hrs. before the autopsy except 7 and 11 days groups in which 131I was injected 24hrs. before the autopsy. Thyroid weight, 131I uptake and conversion ratio were calculated as stated above. T4 group Twenty eight animals received a single injection of T4(10ƒÊg)intraperitoneally and 19 animals served as control. Twenty ƒêc of radioiodine were injected intraperitoneally 24hrs. before the autopsy. At 1, 2, 3, 4, 7 and 11 days after T4 injection, animals were anesthetized with ether and blood was collected from the jugular vein. Thyroid weight, thyroidal 131I uptake and conversion ratio were measured similarly. RESULTS 1)Thyroid function after the withdrawal of MTU The effects of a single injection of 40mg MTU on thyroid function are shown in Table 1 and Figures 1, 2 and 3. Thyroidal 131I uptake was markedly Table 1. Effect of single injection of MTU on thyroid function Results are expressed as mean }standard error of the mean. Forty mg MTU injected intraperitoneally. 131I was of were injected 6hrs. before the 6hrs. group, 12hrs. in 12, groups, and autopsy in before 24 and 48hrs. 24hrs. before in 7 11 groups. and

3 Sept REBOUND IN THYROID AND NEUROSECRETION suppressed as early as 6hrs. following the injection and this marked suppression continued at least for a few days. Although exact measurement has not been made between 2 and 7 days, the effect of MTU to suppress the uptake seemed to disappear gradually, giving normal values of uptake towards the 7th day. This assumption seems probable, since in another occasion the injection of 10mg Fig. 1. Thyroidal 131I uptake after MTU single injection. Plot of data of Table 1. Forty mg of MTU (Methylthiouracil)were injected intraperitoneally. The vertical bars represent the standard error of the means. The same marks are used in the following figures. Fig. 2. Conversion ratio after MTU single injection. Plot of data of Table 1. Forty mg of MTU were injected intraperitoneally. Fig. 3. Thyroid weight after MTU single injection. Plot of data of Table 1. Forty mg of MTU were injected intraperitoneally. Fig. 4. Thyroidal 131I uptake after MTU withdrawal. Graphic plot of data of Table 2. MTU (40mg)was injected for 5 days. Fig. 5. Conversion ratio after MTU withdrawal Graphic plot of data of Table 2. MTU (40mg)was injected for 5 days. Fig. 6. Thyroid weight after MTU withdrawal. Graphic plot of data of Table 2. MTU (40mg)was injected for 5 days.

4 190 SHIMODA Vol.7, A D B E C F G Fig. 7. The accumulation of neurosecretory material in the neurohypophysis after MTU withdrawal. Histological sections was stained with CH and phloxine method. A. 1 day after MTU withdrawal. The material is relatively increased in comparison with control(g). B. 2 days after MTU. C. 3 days after MTU. D. 4 days after MTU withdrawal. In these 3 groups, the amounts of the material in the neurohypophysis is less than that of 1 day, but slightly greater than control(g). E. 7 days after MTU withdrawal. The amounts of this material are much more greater than control(g). F. 11 days after MTU withdrawal. The material decreases significantly under the control range. G. Control. No.3

5 Sept REBOUND IN THYROID AND NEUROSECRETION Fig. 8. Thyroidal 131I uptake after T4 single injection. Graphic plot of data of Table 3. Ten Đg of T4 were injected intraperitoneally. Fig. 9. Conversion ratio after T4 single injection. Graphic plot of data of Table 3. Ten Đg of T4 were injected intraperitoneally. Fig. 10 Thyroid weight after T4 single injection. Graphic plot of data of Table 3. Ten Đg of T4 were injected intraperitoneally. Table 2. Rebound phenomena in thyroid function after MTU withdrawal Results are expressed as mean }standard error of the mean. Forty mg of MTU were injected daily for 5 days. 131I was injected 24hrs. before the autopsy. mercaptoimidazole for 2 days did not produce a rebound in the uptake. Suppression and subsequent recovery of conversion ratio from MTU suppression were very similar to those of 131I uptake. Thyroid weight, however, did not show any

6 SHIMODA Vol.7, No.3 significant change throughout the experimental period. These results thus indicated that no rebound phenomenon was present in any of the 3 thyroid parameters after single injection of MTU. MTU was, therefore, injected for 5 days to produce a clear cut rebound in the following experiment. In contrast to the experiment of a single injection, changes of 131I uptake, conversion ratio and thyroid weight behaved quite differently in animals injected with MTU for 5 days. Thyroidal 131I uptake, which was markedly suppressed on the 1st day, increased gradually and was twice as much as normal at the 7th day (Table 2 and Fig. 4). Thereafter it decreased and reached the normal range at the 11th day. Conversion ratio did show the same pattern as that of the uptake (Table 2 and Fig. 5). Increased thyroid weight was clearly demonstrated at the 1st day, decreasing gradually thereafter, reaching normal range at the 11th day, although thyroid weight at the 2nd day varied rather irregularly(table 2 and Fig. 6). This experiment was repeated to certify the result at the 2nd day, and again somewhat lower value was obtained. 2) Neurosecretory substance in the neurohypophysis after MTU withdrawal Various sizes of droplets of chrome-alum hematoxylin(ch) positive materials were scattered throughout the neurohypophysis of normal animals(fig. 7G). As far as the result of this experiment was concerned, only minor variations have been noted in the amounts of neurosecretory substance in the normal control group. This material of hypothalamic origin, however, clearly increased in size and number at the 1st day in the neurohypophysis of animals which received MTU for 5 days(fig. 7A). It was of interest to observe that the amounts of this material increased further at the 7th day(fig. 7E), and then decreased to a slightly lower level than the control animals(fig. 7F). Thus the changes in the amount of accumulated neurosecretory material were very similar to those of thyroidal 131I uptake and conversion ratio after MTU withdrawal. 3) Thyroid function after T4 withdrawal The effect of a single injection of 10ƒÊg of T4 on thyroidal 131I uptake is shown in Table 3 and Figure. 8. Pronounced suppression was produced with nadir on Table 3. Rebound phenomena after single thyroxine injection Results are expressed as mean }standard error of the mean. Ten ƒêg of T4 were injected intraperitonealty

7 REBOUND IN THYROID AND NEUROSECRETION the 3rd day. Thereafter there was a fairly rapid return to control level. The uptake on the 7th day was almost twice as much as the control and then returned towards the control. Control group was not made in each of the experimental groups, but the uptake was fairly constant in 3 control groups. Almost similar pattern was also noted in the conversion ratio after single injection of 10ƒÊg of T4, although the maximum value obtained at the 7th day was rather lower than that of the uptake(table 3 and Fig. 9). The minimum was reached on the 3rd day and the maximum on the 7th day. Changes in thyroid weight after a single injection of 10ƒÊg of T4 are shown in Table 3 and Figure 10. A similar pattern to those observed with 131I uptake and coversion ratio was observed, but the fluctuation was much lesser than that of the uptake. DISCUSSION Marked increase of thyroidal radio-iodine uptake occurring after goitrogen withdrawal was clearly shown by D'Angelo and his associates(1954). Similar result was reported after the withdrawal of thyroxine in human as well as in experimental animals(riggs et al., 1945; Yamada and Greer, 1959). These results have generally been explained as a compensatory mechanism acting to maintain a constant level of circulating thyroid hormone and are called "rebound phenomena". However, the question arises as to whether the rebound phenomena produced by different thyroid depressant such as goitrogen or thyroxine are essentially the same or different to some extent, since the site and mode of action of those 2 drugs are quite different. The data presented above clearly indicate that rebound phenomena have occurred in both thyroidal 131I uptake and conversion ratio but not in the thyroid weight after the withdrawal of chronic MTU treatment. This not only con- further evidence which might indicate similar rebound in organic binding and release of organic iodine compounds. Although D'Angelo (1954) observed only the rebound in iodine uptake without any concomitant changes in iodine metabolism, it is not surprising to observe the rebound in conversion ratio in this experiment, since iodine uptake in general is the result of the complex process of iodine trapping, organic binding, and release of organic iodine compounds. On the other hand, as shown in Figures 8, 9 and 10, the rebound was noted in iodine uptake and conversion ratio as well as in thyroid weight after release from thyroxine. This difference in thyroid weight was of considerable interest in view of the overall mechanism of the rebound occurring after release from thyroid depressant. Although the measurement of circulating TSH was not made in this experiment, it may be postulated that so-called" rebound phenomena" and different response in thyroid weight are related to changes of circulating TSH. This speculation was illustrated in Figure 11. Blood concentration of TSH in MTU group may be elevated at the beginning of the experiment, and was followed by gradual decrease without showing any rebound. At the 7th day

8 SHIMODA Vol.7, No.3 Fig. 11 Graphic representation of possibie relationship among several factors which may be responsible for the production of rebound phenomena in thyroid parameters after release from MTU(A) or T4(B). where marked rebound was observed in the uptake and conversion ratio, TSH in the blood might still be higher than normal and goitrogen in the thyroid as well as in the blood was washed out completely by that time, Thus the rebound in

9 REBOUND IN THYROID AND NEUROSECRETION 131I uptake and conversion ratio was produced, but not in thyroid weight. While, TSH in the blood of T4 group might be very low in the beginning of the experiment and was followed a gradual increase. At the 7th day TSH might exceed the normal and thus rebound phenomena were produced in all thyroid parameters. Since it is well established that the hypothalamus regulates pituitary TSH secretion(harris, 1955;Greer, 1957; Yamada, 1959), the participation of the hypothalamus might be expected for the production of this rebound phenomena. Although the exact proof is lacking, hypothalamic control of the pituitary TSH secretion is mediated through some neurohumoral substance(s) which is carried by the portal vessels down to the pituitary(harris, 1956; Nikitovich-Winer and Everett, 1958). It is known that neurosecretory activity in the hypothalamus changes in response to thyroid activity(shiozaki, 1956; Shimizu, 1959; Shichijo et al., 1959)and the neurosecretory material does actually enter the hypophyseal portal vessels(bargmann, 1954; Scharrer and Scharrer, 1954; Hild, 1956). Further it is sometimes claimed that so-called posterior hormone seems to stimulate TSH secretion (Froja and Martini, 1953;Dubreuil and Martini, 1956; Botali, 1957). The pronounced rebound of neurosecretory material in the neurohypophysis thus seems very interesting in this connection. It should be mentioned here that TSH rebound in the adenohypophysis did occur after goitrogen withdrawal(d'angelo, 1959). The different pattern in the decrease of synthesis and of release of neurosecretory material might possibly be the cause in producing the rebound in the neurohypophysis as well as postulated in TSH rebound of the anterior pituitary. It seems unlikely that this rebound in the neurosecretory system is an accidental occurrence and has no relationship with the rebound of pituitary-thyroid system occurring after release from chronic goitrogen treatment. Although it is premature to draw definite conclusion from this observation, these similarities in the rebound of anterior and posterior hormones lead to speculate the possible functional relationship between the anterior pituitary and the hypothalamus, and provide a promising starting point for further studies. SUMMARY Comparison of the effects of MTU and T4 withdrawal on different thyroid parameters has been made in the rat. After the withdrawal of chronic MTU treatment, marked rebound in thyroidal 131I uptake and conversion ratio was produced but not in thyroid weight, while no rebound was produced in any of thyroid parameters after a single injection of MTU. After a single injection of T4, however, pronounced rebound was produced in thyroidal 131I uptake, conversion ratio and thyroid weight. A similar type of rebound of neurosecretory substance was also produced after the withdrawal of chronic MTU treatment. It is postulated that the difference in the response of thyroid weight might be related to different pattern of circulating TSH. Possible participation of hypothalamic neurosecretory system for the production of rebound phenomena is discussed.

10 ACKNOWLEDGEMENT The author wishes to express his hearty thanks to Prof.K.Shichijo, Gunma University, for his kind direction, encouragement and criticism in this study. REFERENCES Azzali, G. and C. Shernin (1959). Bull. soc. ital. biol. sper. 34, 109. Bargmann, W. Das Zwischenhirn-Hypophysensystem Springer, Berlin (1954). Bogdanove, E. M. and N. S. Halmi (1953). Endocrinology 53, 274. Botali, P. M.(1957). Ciba Foundation Colloquia on Endocrinology 11, 52. Brown-Grant, K.(1957). Ciba Foundation Colloquia on Endocrinology 10, 97. D'Angelo, S. A., C. E. Stevens, K. E. Paschkis, A. Cantarow, F. W. Sundermann and G. Friedler (1954). Endocrinology 54, 565. D'Angelo, S. A.(1954). Brookhaven Symposia in Biol. 7, 9. D'Angelo, S. A. The Endocrine Society, Program of the 41st Meeting p.50 (1959). Dougherty, J., J. Gross and C. P. Leblond (1951). Endocrinology 48, 700. Dubreuil, R. and L. Martini. XXth International Congress of Physiology p.257 (1956). Froja, A. and L. Martini (1953). Arch. intern. pharmacodynamie 93, 167. Ganong, W. F., D. S. Frendrickson and D. M. Hume (1955). Endocrinology 57, 355. Goldberg, R. C., J. Wolff and R. O. Greep (1956). Endocrinology 60, 38. Greer, M. A.(1957). Recent Progr. in Hormone Research 13, 57. Harris, G. W. Neural Control of the Pituitary Gland, Edward Arnold Ltd, London. p.132 (1955). Harris, M. A. Hypothalamic-Hypophyseal Interrelationships. Charles C. Thomas Publisher, Springfield, Illinois p.31 (1956), Harris. G. W. and J. W. Woods (1958). J. Physiol. 143, 246. Herring, P. T.(1908). Quart. J. Exptl. Physiol. 1, 281. Hild, W. Hypothalamic-Hypophyseal Interrelationships. Charles C. Thomas Publisher, Springfield, Illinois p.17 (1956). Hoskins, R. C.(1949). J. Clin. Endocrinol. 9, Nikitovich-Winer and J. W. Everett (1958). Endocrinology 63, 961. Riggs, D. S., E. B. Man and A. W. Winker (1945). J. Clin. Invest. 24, 722. Scharrer, E. and B. Scharrer (1954). Recent Progr. in Hormone Research 10, 184. Shibusawa, K., S. Saito, K. Nishi, T. Yamamoto, C. Abe and K. Tomizawa (1956). Endocrinol. Japon. 3, 139. Shichijo, K., S. Shimoda and T. Shimizu (1959). Gunma J. Med. Sci. 3, 281. Shiozaki, N.(1956). Endocrinol. Japon. 3, 242. Shimizu, T.(1959). Endocrinol. Japon. 6, 75. Yamada, T.(1957). Endocrinol. Japon. 4, 110. Yamada, T.(1959). Endocrinology 65, 216. Yamada, T. and M. A. Greer (1959). Endocrinology 46, 559.