For studying the relationship between the pineal gland and hypophysial prolactin (PRL) secretion, the experiment was done as follows :
Exp. 1. The effect of a pinealectomy on serum and pituitary PRL levels.
Adult female Wistar rats were divided into three groups. Group 1 : The rats underwent pinealectomy or a sham-operation by the Kuszak and Robin modified method 3 weeks after castration. They were sacrificed by decapitation 9 days after the pinealectomy. Group 2 : The rats underwent pinealectomy or a sham-operation 3 weeks after castration and were injected with estrogen-progesterone 1 week after the pinealectomy. They were sacrificed by decapitation 48 hrs after the injection. Group 3 : The rats underwent pinealectomy or a sham-operation on the 15-17th day of pregnancy. After delivery, on the third day post partum, the litter was separated to avoid the influence of suckling for 30 min, and they were sacrificed by decapitation.
Exp. 2. The effect of melatonin, serotonin and arginine vasotocin (AVT) on serum and pituitary PRL levels.
Melatonin, serotonin and AVT, in doses of 50,100 and 200μg, were injected i.v. into Group 2 and Group 3 described in Exp. 1. above. After the results of the preliminary experiment, the rats were decapitated at 30 min after the administration.
The experimental conditions were as follows : room temperature; 22±2°C, light : dark; 12 : 12 hrs, maintained ad-libitum. The decapitations were done between 3 : 00 pm - 5 : 00 pm. Prolactin levels in the serum and pituitary were determined by radioimmunoassay.
Pinealectomy made serum PRL levels increase significantly and pituitary PRL levels decrease significantly in the castrated (Group 1) and puerperal (Group 3) rats. On the other hand, serum PRL levels were significantly decreased by pinealectomy in the castrated estrogen-progesterone primed (Group 2) rats compared with the control rats (sham-operated). After the i.v. injection of melatonin 50μg, serum PRL levels deceased below preinjection levels in the pinealectomized rats of both Group 2 and Group 3. Namely, the serum PRL levels in the non pregnant rats were significantly more depressed. On the other hand, the serum PRL levels of the puerperal rats, increased by pinealectomy, were also depressed by a melatonin 50μg injection.
In the sham-operated rats, both Group 2 and Group 3, a melatonin 50μg injection increased the serum PRL levels, especially in the puerperal rats (Group 3). On the other hand, the serum PRL levels did not decrease with AVT and serotonin injections in either group of pinealectomized rats. AVT administration made serum PRL increase greatly in the pinealectomized and sham-operated rats. There was no significant difference on the serum PRL increased by AVT between the pinealectomized and sham-operated rats. The hypophysial PRL levels were greatly increased by melatonin after a 50μg injection in the pinealectomized and sham-operated puerperal rats.
These results of our experiments, judging by the changes in the levels of serum and pituitary PRL, may be elucidated as follows :
a) In non pregnant rats, the pineal gland probably acts to stimulate PRL release by inhibiting hypothalamic PIF. On the other hand, in puerperal rats, the pineal gland probably acts to inhibit PRL release by inhibiting hypothalamic PRF.
b) The physiological effect of melatonin on PRL secretion, within a physiological range of 50pg melatonin, will indicate the releasing and inhibiting action. The effect of melatonin on hypophysial PRL might be an inhibiting action, judging from the depressed serum PRL levels resulting from a 50μg melatonin injection into the pinealectomized rats. However, in the presence of the intact pineal gland, the PRL releasing effect of melatonin -possibly by releasing AVT in the pineal gland -might be superior to the PRL inhibiting effect of melatonin alone.

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SP₁ (pregnancy specific β₁-glycoprotein) was purified from human placentae, and the metabolism of ¹²⁵I labeled SP₁ was studied in non-pregnant and pregnant mice of ICR strain.
Purification of SP₁ was carried out by the method of Bohn (1971) and Nose (1981) with modifications. The procedure was as follows : Placental tissues were homogenized with an equal volume of 0.4% NaCl and centrifuged to remove the debris. Into this supernate, rivanol was added to 0.8% concentration. Then the solution was centrifuged, and into the supernate thus obtained, solid ammonium sulfate was added to 50% saturation. The precipitate was thoroughly dialysed against 0.01M tris-HCl buffer pH 8.0 and was applied to the column of DEAE-cellulose equilibrated with the same buffer. After washing, the proteins were eluted with 0.02M tris-HCl buffer pH 8.0 containing 0.45M NaCl. These proteins, concentrated by 503o saturated ammonium sulfate, were applied to a Sephadex G-150 column and eluted with 0.05M tris-HC1 buffer pH 8.0 containing 0.02M NaCl. The SP₁ containing fraction was applied to a DEAE-Sephadex A-50 column, and the elution was carried out with a NaCl gradient (from 0.2% to 2%) in 0.01M tris-HC1 buffer pH 7.6. Next, concentrated SP₁ rich fractions were applied to a Sephacryl S-200 column and eluted with 0.05M tris-HCl buffer pH 8.0 containing 0.5M NaCl.
The SP₁ fractions were applied to an anti SP₁ bound Sepharose 4B column and were eluted with 0.2M glycine-HCl buffer pH 2.5. Each fraction was immediately neutralized by 1.0M tris to pH 7.6. After having been dialysed against distilled water, the pooled protein fraction was lyophilized.
By immunoelectrophoresis the obtained SP₁ formed one precipitated line using anti pregnant serum as well as and SP₁ serum, and did not react with any and sera except for anti SP₁ by the Ouchterlony method. Furthermore, polyacrylamide gel electrophoresis showed one main band, and the purity was estimated approximately 98% from a densitometric pattern.
A preparation of purified SP₁ was labeled with ¹²⁵I by the Chloramine-T method. Radio-iodinated SP₁ was separated from the reaction mixture by gel filtration on a Sephadex G-50 column using 0.01M sodium phosphate buffer pH 7.4 added 1% bovine serum albumin. The radioactivity of each fraction was measured in a γ-spectrometer. The free radioactive iodine of the fractions which reacted with anti SP₁ serum was less than 1%. These fractions were pooled and stored at -20°C until use.
¹²⁵I-SP₁ (0.1ml of approximately 2.5 × 10⁶ C.P.M./ml radioactivity) was injected into the subcutaneous tissue of both non-pregnant and pregnant mice. The experiment was carried out in 6 non-pregnant mice and 4 pregnant mice as one group. The animals were killed at 0.5, 1, 2, 4, 8 and 16 hours after the injection. The organs and tissues were collected, weighed and analysed for accumulated radioactivity.
Non-pregnant mice tissue radioactivity exhibited the highest count in the kidney, followed by blood, ovary & uterus, spleen, liver, muscle and fat at 30 min. after the injection; similar tendencies were observed at other times. Radioactivity of the kidney was more than twice that of blood. A blood disappearance curve showed a half life of 8 hours.The radioactive substance was excreted in urine at a very high level from 30 min. to 4 hours, and in the faeces, it peaked at 8 hours. Pregnant mice tissue exhibited the highest count in the kidney, followed by blood, spleen, ovary & uterus, placenta, fat, muscle and fetus at 30 min. A blood disappearance curve showed a half life of about 11 hours. Radio-activity of the placenta exceeded that of blood after about 1.5 hour from the injection and reached a peak at 4 hours.

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In order to clarify the role of dopamine in aldosterone secretion, the effect of metoclopramide, a dopamine antagonist, on plasma aldosterone concentration was evaluated in five normal subjects and five patients with primary aldosteronism, to whom 2mg/day of dexamethasone was administered for 2 days to eliminate the influence of ACTH prior to the study.
The dexamethasone treatment slightly decreased plasma aldosterone concentrations (PAC) from 14.2 ± 1.8 (mean ± s.e.) to 12.9 ± 2.3ng/dl in normal subjects and also from 36.9 ± 15.6 to 35.1 ± 16.7ng/dl in patients with primary aldosteronism. Plasma cortisol concentrations (PF) were markedly decreased by dexamethasone administration from 6.1 ± 2.1 to 1.1 ± 0.1μg/dl (p<0.001) in normal subjects and from 7.1 ± 1.8 to 0.7 ± 0.1 μg/dl (p < V0.001) in patients with primary aldosteronism, while plasma renin activity (PRA) and plasma prolactin concentrations (PRL) did not charge with the dexamethasone administration in either normal subjects or patients with primary aldosteronism.
An i.v. bolus injection of metoclopramide (10mg) rapidly increased PAC from 12.9 ± 2.3 to 17.3 ± 3.3ng/dl (p < 0.05) in normal subjects and from 35.1 ± 16.7 to 38.5 ± 17.0 ng/dl (p < 0.05) 5 min after the administration in patients with primary aldosteronism. PAC elevated maximally to 23.8 ± 3.4ng/dl (p < 0.02) in normal subjects and to 45.1 ± 16.7 ng/dl (p < 0.02) in patients with primary aldosteronism 30 min after the administration of metoclopramide. There were no significant differences in change of PAC in response to metoclopramide between normal subjects and patients with primary aldosteronism. PRL elevated significantly from 4.5 ± 1.1 to 102.0 ± 9.3ng/ml (p < 0.001) in normal subjects and also from 8.7 ± 3.9 to 108.4 ± 23.1ng/ml (p < 0.01) in patients with primary aldosteronism 30 min after the administration. PRA, PF, serum potassium and sodium concentrations did not significantly change with the metoclopramide administration in either normal subjects or patients with primary aldosteronism. There was no significant relationship between the increase in PAC and PRA and the increase in PAC and PRL with metoclopramide in either normal subjects or patients with primary aldosteronism.
These results suggest that metoclopramide acts directly upon the adrenal cortex to stimulate the secretion of aldosterone, and that aldosterone secretion may be suppressed by dopamine in primary aldosteronism as well as in normal subjects.

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A 54-year-old man was admitted to our hospital on July 18, 1980. His familial history showed no hypertension and his past medical history was not significant. At the age of 39, hypertension (220/-mmHg) was first diagnosed, but it was not improved by antihypertensive therapy. Moreover, at the age of 53, hypertension (220/110 mmHg), hypokalemia, low plasma renin activity (PRA) and significant high plasma aldosterone concentrations (PAC) were indicated.
Laboratory findings disclosed hypokalemia (2.9 mEq/l) and metabolic alkalosis. A high PAC of 20.4 ng/dl and a low PRA of 0 ng/ml/h were detected. PRA was suppressed to 0 ng/ml/h even after quiet ambulation for 2 hours with the administration of 1 mg/kg furosemide. Urinary excretion rates of 17-OHCS and 17-KS were within normal limits. Retroperitoneal pneumography, computed tomography and adrenal scintiscan all failed to detect adrenal adenoma, but a right adrenal adenoma was suspected with the determination of PAC in the renal veins. The pathological findings of the excised adrenal gland showed hyperplasia of the zona glomerulosa and the subglomerulosa. After the adrenalectomy, the hypertension and hypokalemia did not improve. Plasma ACTH was within normal limits at 81 pg/ml. Relatively high levels of plasma deoxycorticosterone, 18-hydroxydeoxycortico-sterone, corticosterone and 18-hydroxycorticosterone were found. Plasma levels of glucocorticoids and androgens were within the normal range, and urinary excretion rates of pregnandiol and pregnantriol were normal. PAC was significantly increased by exogenous ACTH administration. Administration of dexamethasone decreased the blood pressure from 210/110 mmHg to 140/80 mmHg and increased the serum potassium from 3.0 mEq/1 to 4.2 mEq/l, the PRA from 0 ng/ml/h to 1.2 ng/ml/h, and the suppressed PAC to 0 ng/dl.
The data suggest that the high level of aldosterone in the patient depended upon the fact that the receptors in the aldosterone-producing cells of the adrenal glands were congenitally sensitive to endogenous ACTH rather than angiotensin II.

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The responses of plasma active and inactive renin to various conditions which influence renin release were studied in normal subjects. Simultaneous measurements of urinary kallikrein and plasma prekallikrein were performed to assess the possible role of renal or plasma kallikrein in the in vivo activation of inactive renin. The effect of aprotinin infusion on plasma active and inactive renin was also examined. Active renin was measured by radioimmunoassay. Inactive renin was calculated as the difference between total renin after trypsin activation and active renin. Both urinary kallikrein and plasma prekallikrein were determined using fluorogenic substrates.
Short term stimulation with furosemide administration and ambulation, infusion of isoproterenol, and administration of captopril increased active renin, but had little effect on inactive renin. Sodium depletion and sodium repletion, each for 4 days, induced parallel changes in active and inactive renin. The administration of propranolol for 4 days decreased active renin but did not change inactive renin. In the furosemide study, the proportion of active renin (active renin/total renin) was significantly correlated with urinary kallikrein.There was a significant correlation between the proportion of active renin and plasma prekallikrein during the long term sodium balance study. Aprotinin infusion for 3 days did not affect either active or inactive plasma renin.
These results suggest that the control mechanisms of active and inactive renin are different. Renal or plasma kallikrein seems to be involved in part in the in vivo activation of inactive renin to the active form. We could not obtain any direct evidence that serine protease might participate in this activation.

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Immunosuppressive acidic protein (IAP) has been reported to be found in the sera of not only cancer patients but also collagen diseases such as SLE and rheumatoid arthritis and certain inflammatory diseases such as liver abcess and pyothorax. IAP is proved to be one of the heterogeneity of human serum α₁ -acid glycoprotein and to suppress both phytohemagglutinin-induced lymphocyte blast formation and mixed-lymphocyte reaction in vitro (Cancer Research, 41 : 3244, 1981).
In the present study, we measured IAP by the single radial immunodiffusion methods in patients with various thyroid diseases.
The IAP concentration in patients with subacute thyroiditis was 956 ± 256μg/ml (mean ± S.D., n=32), which is significantly higher than that in hyperthyroidism (267 ± 64μg/ml, n=10), hypothyroidism (328 ± 81μg/ml, n=9), chronic thyroiditis (366 ± 120 μg/ml, n=24) and destructive thyroiditis (352 ± 34μg/ml, n=4), respectively. The normal value of IAP concentration is below 500μg/ml.
After therapeutic administration of prednisolone or salicylate to patients with subacute thyroiditis, IAP rapidly decreased to normal levels in association with the recovery of elevated erythrocyte sedimentation rate (ESR) and serum thyroxine (T₄) and triiodothyronine (T₃).
In the densitometric tracing of IAP separated by thin-layer isoelectric focusing at a pH range of 2.5 to 5, the major acidic protein had an isoelectric point (pI) of 3.0 in patients with subacute thyroiditis during the active phase as well as in one patient with gastric cancer. Furthermore, the acidic protein at pI 3.0 decreased and the one at pI 3.1, which is the major protein observed in normal serum, became prominent during the recovery phase in patients with subacute thyroiditis. The major acidic protein also had a pI of 3.1 in the sera of patients with hyperthyroidism, hypothyroidism and chronic thyroiditis.
IAP remained within the normal range in a patient with destructive thyroiditis who showed thyrotoxic symptoms and then became hypothyroid.
It is suggested that the determination of IAP is useful in the clinical diagnosis of subacute thyroiditis. Further analysis of IAP might be helpful for the clarification of the pathogenesis of subacute thyroiditis.

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