The Japanese Journal of Physiology 19, pp.465-476, 1969

EFFECTS OF , , 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 . 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 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- (L-T3), tetraiodothyroacetic acid (TA4), monoiodotyrosine (MIT), diiodotyrosine (DIT), corticosterone, and 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 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 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. These tATPase properties are similar to those of tATPase from rat brain and pig thyroid.12) The specific activity of the anterior pituitary tATPase preparation from intact rats was 3 times greater than that of pig thyroid tATPase and less

than 1/5 that of rat brain tATPase. The tATPase of rat anterior pituitary was activated by thyroidectomy.

This activation was depressed by T4 injected at a low dose of 20ƒÊg/100g body weight in a short time of 4 hr after injection. The effect was also observable with the preperation from intact rats. The marked depressing effect of T4 was also observable in vitro. L-T4, L-T3 and TA4 exerted their depressing effect even at a concentration as low as 5•~10-8M. In contrast, physiologically inactive iodide compounds, such as D-T4, DIT, MIT and KT, showed no effect even at 5•~10-6M.

The results were similar with the adrenal cortical hormones. Adrenale- ctomy caused activation. Injection of corticosterone (1mg/100g body weight) depressed the enzyme derived from both intact and adrenalectomized rats.

Glucocorticoids-corticosterone, cortisone, cortisol and dexamethasone-were also effective in vitro even at the level of 10-8M.

All these facts strongly suggest one of the mechanisms by which the thyroid and the adrenal inhibit the function of the anterior pituitary in the manner of so-called negative feedback. If the anterior pituitary tATPase plays a role in transport of amino acids, it would be possible to explain the depression of amino acid transport by T415) and glucocorticoide from depres- sion of this enzyme by these hormones. This assumption is however, not tenable because it was found that transport of amino acid into the anterior pituitary is entirely insensitive to ouabain.4) Therefore depression of tATP- ase by T4 and glucocorticoids may have nothing to do with the mechanism of depression of amino acid transport by T4 and glucocorticoids. We only 474 I. TAKAGI AND K. YAMAMOTO

tentatively suppose that stimulation of pituitary tATPase by thyroidectomy or adrenalectomy, and inhibition of this enzyme activity by thyroid hormones and glucocorticoids may cause certain changes in ionic balance of Na+and + inside and outside the anterior pituitary cells , and these changesK may have a certain connection with the mechanisms of negative feedback regula- tion of the anterior pituitary functions. Effectiveness of thyroid and adrenocortical hormones in vivo after a short time lag of a few hours and in vitro at concentrations close to physiological level indicates that these hormones directly depress tATPase of rat anterior pituitary.Therefore, tATPase can be called one of the target enzymes of hormones, of which only a few, such as adenyl cyclase,11) are known. It is difficult to understand the fact that tATPase from different tissues, which has apparently similar properties, is quite different in susceptibility to hormones. The enzyme derived from rat anterior pituitary was depressed specifically by thyroidal and adrenocortical hormones, but not by other steroid hormones. The enzyme of pig thyroid was only sensitive to estrogens, not to adrenal cortical hormones, androgens and thyroid hormones. The enzyme from rat brain was affected by none of these hormones. This kind of thing is generally ascribed to so-called tissue specificity but we have no explana- tion about it. M++-ATPase was different from tATPase in response to hormone admi- nistration. The former enzyme was activated by either thyroidectomy or adrenalectomy, but neither injection in vivo nor addition in vitro of thyroidal or adrenocortical hormones did change the activity of the enzyme obtained from intact, thyroidectomized or adrenalectomized rats. Therefore, Mg++- ATPase may not be a target enzyme of these hormones, and activation caused by thyroidectomy or adrenalectomy may be an indirect effect of the lack of these hormones.

SUMMARY

The activity of Nat+, K+-activatied ATPase or transport ATPase (tATP-

ase) was measured on a cell membrane fraction of the anterior pituitary of

male rats. The enzyme activity increased either 2 weeks after thyroidectomy

or one week after adrenalectomy. The increased activity was depressed 4

hours after injection of a small amount of T4 (20ƒÊg per 100g body weight) or corticosterone (1 mg per 100g body weight), respectively. Similar depres-

sive effects were observed with the enzyme preparation from intact animals.

Physiologically occurring thyroid hormones, T4, T3 and TA4, depressed the

enzyme in vitro. Glucocorticoids, corticosterone, cortisol, cortisone and dexa- methasone, were similarly effective in vitro. The effective concentrations of these hormones were as low as 10-8M. In contrast, D-T4, iodide, iodotyrosines, PITUITARY T-ATPASE AND THYROID & ADRENAL 475 testosterone propionate, estradiol-17ƒÀ and cholesterol were entirely ineffective.

These results indicate that rat anterior pituitary tATPase is one of the target enzymes of the thyroid and adrenocortical hormones. The enzyme is directly andspecifically depressed by these hormones.

These findings were discussed in relation to the mechanisms of the feedback regulation of anterior pituitary functions by the thyroid or adrenal cortex.

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FOOT NOTE

Trivial names and abbreviations used:

T4 = thyroxine;

T3 = triiodothyronine; TA4 = tetraiodothyroacetic acid;

MJT = monoiodotyrosine; DIT = diiodotyrosine;

dexamesasone = 9 ƒ¿-fluoro - 11 / ƒÀ, 17, 21-trihydroxy- 16 ƒ¿-methylpregna- 1, 4-diine-3, 20-dione.