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EFFECTS of THYROIDECTOMY, ADRENALECTOMY, THYROID HORMONES and GLUCOCORTICOIDS on Na+, K+-ACTIVATED ATP-ASE of RAT ANTERIOR PITUITARY

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EFFECTS of THYROIDECTOMY, ADRENALECTOMY, THYROID HORMONES and GLUCOCORTICOIDS on Na+, K+-ACTIVATED ATP-ASE of RAT ANTERIOR PITUITARY The Japanese Journal of Physiology 19, pp.465-476, 1969 EFFECTS OF THYROIDECTOMY, ADRENALECTOMY, THYROID HORMONES AND GLUCOCORTICOIDS ON Na+, K+-ACTIVATED ATP-ASE OF RAT ANTERIOR PITUITARY Isao TAKAGI AND Kiyoshi YAMAMOTO Department of Physiology, Institute of Endocrinology, Gunma University, Maebashi It is well known that the function of the anterior pituitary is controlled by hormones secreted from several peripheral endocrine glands. In the pre- vious reports from this laboratory, TONOUE and YAMAMOTO 14-18)and HOSHINO et al.2, 3) showed that transport and incorporation of amino acids were stimu- lated in rat anterior pituitary after either thyroidectomy or adrenalectomy. Injection of thyroxine (T4) or glucocorticoids to thyroidectomized or adrena- lectomized rats was found to depress the increased transport and incorporation. In vitro addition of these hormones was also highly effective in depressing these two activities. Since Na+, K+-activated ATPase or transport ATPase (tATPase) has been reported to participate in amino acid transport in several tissues 1,5, 7, 8, 19, 20), it was of interest to investigate changes in pituitary tATPase activity after thyroidectomy, adrenalectomy or administration of thyroid hormones or gluco- corticoids in vivo and in vitro. Another purpose of this experiment was to compare the effects of these hormones on tATPase of the anterior pituitary and the thyroid. Thyroidal tATPase was stimulated specifically by estrogens but was not sensitive to either thyroid or adrenocortical hormonesi 12). A part of the present results has been reported preliminarily 13). MATERIALS AND METHODS Adult male rats of Wistar strain, weighing 150 to 200g, were kept on pellets (Oriental Co.) and water ad lib. in a room of a constant temperature (24•}2•Ž). Thyroidectomy was performed surgically two weeks and adrenaletomy one week be- fore the animals were used for experiments. Adernalectomized animals were maintained on drinking water which contained 1% NaCl. For one series of experiment, usually 12-20 anterior pituitaries were pooled, homo- Received for publication May 2, 1969 高 木 勲,山 本 清 465 466 I. TAKAGI AND K. YAMAMOTO genized with 10 ml ice-cold distilled water brought to pH7.5 by the addition of 1 mM NaHCO3. Pituitary cell membrane was prepared as was described before .n) The final preparation was resuspended in 3.4-4.5ml distilled water (protein concentration , 0.4-0.7mg/ml) and used for ATPase assay. The reaction mixture and assay proce- dures were the same as described before .12) ATPase activity was assayed both in the presence of Mg++, Na+ and K+ (total ATPase) and in the presence of Mg++ only (Mg++- ATPase). The difference between these two assays showed tATPase activity. Measu- rement was made in duplicate, and the mean values, which were very close each other, were shown in each of the following data. Each series of experiment was carried out at least three times for confirmation of the result . Protein content of the enzyme preparation was determined by the method of LOWRY at al.6) Tris-ATP was prepared from commercial potassium-ATP (Sigma Chem . Co.) by the method of SCHWARTZ et al.9) Ouabain, L-T4, D-T4, L-triiodothyronine (L-T3), tetraiodothyroacetic acid (TA4), monoiodotyrosine (MIT), diiodotyrosine (DIT), corticosterone, cortisone and cortisol were obtained from Sigma . Co. Estradio1-173 was generously supplied by Teikokuzoki Pham. Co. Dexamethasone was purchased from Merk Co. and testosterone from Chemical Specialities Co. RESULTS tATPase of rat anterior pituitary FIG.1 shows the tATPase activity in the presence of Na+ and K+ at various combinations. The highest activity was obtained at a combination of 100 mM Na+ and 20 mM K . This com- bination of Na+ and K+ was used throughout the following experiments . FIG.2 indicates the inhibition by ouabain. A half-maximal inhibition FIG.1. The effect of varying Na+ and K+ concentrations on the ATPase activity of pituitary preparation. PITUITARY T-ATPASE AND THYROID & ADRFNAL 467 FIG.2. Inhibition of tATPase activity by varying concentration of ouabain. FIG.3. Effect of pH on both Me++-ATPase and tATPase activities. ●― ● tATPase of the intact rats ○― ○ Mg++-ATPase without Na+ and K+ of the intact rats ■― ■ tATPase of the thyroidectomized rats □― □ Mg++-ATPase of the thyroidecto- mized rats ▲― ▲ tATPase of the adrenalectomized rats △― △ Mg++-ATPase of the adrenalecto- mized rats 468 I. TAKAGI AND K. YAMAMOTO was observed at concentration between 10-7M and 10-6M, and the complete inhibition at 10-4M ouabain. FIG. 3 shows tATPase activity at pH's ranging from 6.5 to 9.0. The optimal pH of the tATPase was around 8.0, and that of Mg++-ATPase around 7.5, either with normal, thyroidectomized or adrenalectomized rats. Effect of thyroidectomy or adrenalectomy FIG. 3 also shows that pituitary ATPase activity increases after thyroidectomy or adrenalectomy. Similar stimulatory effect of thyroidectomy is seen in experiments listed in TABLE1. Although the extent varied from experiment to experiment, this stimulation was reproducible. The percent increase of tATPase were 49 to 128. Mg-+- ATPase was also activated (57-76%) by thyroidectomy. TABLE 2 shows the stimulatory effect of adrenalectomy. Percent increase of tATPase was 41 to 110, that of Mg++-ATPase being 42-72. TABLE1. Effect of thyroidectomy on anterior pituitary ATPase of the rat * Percent increase TABLE2. Effect of adrenalectomy on anterior pituitary ATPase of the rat * Percent increase PITUITARY T-ATPASE AND THYROID & ADRENAL 469 Effect of T4 and other iodide compounds in vivo and in vitro TABLE 3 shows the effect of T4 (20ƒÊg/100g body weight) injected to intact or thyroide- ctomized rats on pituitary tATPase. With both groups, the enzyme activity began to decrease 4hr (9-24%) after the injection, and the depression in- creased after 8 (31-43%) and 16hr (43-83%). After 16hr, the effect was more marked with thyroidectomized rats. On the contrary, the depressive effect was not clear on Mg++-ATPase. TABLE3. Depression of anterior pituitary tATPase by T4 injection TABLE4. Depression of anterior pituitary tATPase by thyroid hormones added in vitro 470 I. TAKAGI AND K. YAMAMOTO TABLE 4 shows that in vitro addition of T4 and other physiologically active thyroid hormones depressed the enzyme from both intact and thyroide- ctomized rats. At concentrations of 5•~10-6M and 5•~10-IM of the hormones, the depression was marked (38-94%). Even at an extremely low concentration of 5•~10-8 M, a slight but consistent depression was observable in this experi- ment and repeated experiments which were not listed here. However, Mg++ ATPase was not affected by these hormones. TABLE 5 indicates that D-T4 and other physiologically inactive iodide codide comrounds-KI, MIT and DIT-were not effective on tATPase. Effect of glucocorticoids in vivo and in vitro TABLE 6 shows the effect of corticosterone (1mg/100g body weight) injected to intact or adrenalecto- mized rats on anterior pituitary tATPase. The enzyme activity began to TABLE5. Anterior pituitary tATPase activity in the presence of DT4, MIT, DIT and iodide in vitro TABLE6. Depression of anterior pituitary tATPase by corticosterone injection PITUITARY T-ATPASE AND THYROID & ADRENAL 471 decrease 4hr (12, 26%) after injection, and the depression increased towards 8 (22, 47%) and 16 hr (47. 52%). Again Mg++-ATPase did not decrease by the hormone injection. TABLE7 indicates the effect of several glucocorticoids added in vitro. Corticosterone depressed the enzyme from both intact and adrenalectomized rats. The other glucocorticoids-cortisone, cortisol and a potent synthetic glucocorticoid, dexamethasone-were all effective in depressing tATPase. Except for dexamethasone, which showed a marked inhibition (-56%) even at 10-8M, the effect of the other corticoids was slight at the same concentra- tion level but this effect was observable consistently in several other additional experiments. Mg++-ATPase activity was not altered at all. In contrast to these glucocorticoids, other steroid compounds, estradiol-17ƒÀ , testosterone propionate and cholesterol, were not effective on tATPase, and also on Mg++-ATPase, in vitro. Effect of thyroid and steroid hormones on rat brain ATPase activity To test whether thyroid, adrenocortical and other steroid hormones are effectve on the same enzyme from a tissue other than the anterior pituitary, tATPase was prepared from rat brain after the method of SKOU,10) and was studied. TABLE7. Depression of anterior pituitary tATPase by glucocorticoids added in vitro 472 I. TAKAGI AND K. YAMAMOTO The specific activity of this tATPase preparation was 5 to 10 times higher than that of the anterior pituitary. The highest activity of the brain tATPase was observed at the same Na+-K+ combination as in the case of anterior pituitary tATPase. The sensitivity of the brain enzyme to ouabain was also similar to that of anterior pituitary tATPase, except for that about 12% of the full-activity was still remaining in the presence of 10-4M ouabain. TABLE8. Anterior pituitary tATPase activity in the presence of testosterone, estradiol and cholesterol in vitro TABLE9. Rat brain ATPase activity in the presence of thyroid and steroid hormones PITUITARY T-ATPASE AND THYROID & ADRENAL 473 TABLE 9 shows no effect of L-T4, MIT, corticosterone, estradiol-17ƒÀ and testosterone propionate on rat brain ATPase in vitro at concentrations of 5•~10-6 to 10-8M. This result indicates a characteristic property of anterior pituitary tATPase that is highly sensitive to thyroid hormones and gluco- corticoids. DISCUSSION The present tATPase preparation of rat anterior pituitary cell membranes had the following properties; The highest enzyme activity was obtained with a combination of 100mM Na•} and 20mM K+. The enzyme was ouabain- sensitive; the half-maximal inhibition was f ound at a concentration between 10-7M and 10-6M, and the complete inhibition at a concentration of 10-4M ouabain. Optimal pH for the tATPase was about 8.0 at the optimal Na+ and K+ combination, while that for Mg++-ATPase was about 7.5.
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