In order to investigate the possible role of mineralocorticoid in the regulation of digoxin-like substance (DLS), 9α-fluorocortisol (9-F) was administered to 6 healthy men and urinary exeretion of DLS was measured. The administration of 0.6 mg of 9-F caused slight increases in body weight and blood pressure and significant decreases in urinary Na excretion, plasma renin activity and plasma aldosterone, which indicate the expansion of extracellular fluid (ECF) volume by 9-F administration. Urinary excretion of DLS decreased significantly from the baseline level of 43.3±2.6 (SEM) to 29.8±5.1 (SEM) ng/day; digoxin equiv. after 9-F. These results suggest that a large dose of mineralocorticoid may suppress DLS despite an increase in the ECF volume.

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The acute effect of a hypoglycaemic dose of 0.5U/100g BW insulin administered intramuscularly on calcium metabolism was investigated in fasted alloxan-treated rats. It was found that the hypercalcaemic effect of insulin was evident only in thyroparathyroidectomized (TPTX) and not in parathyroidectomized (PTX) rats. A subcutaneous administration of 180 MRC mU/100g BW calcitonin abolished the calcium raising effect of insulin in TPTX rats suggesting a protective role of calcitonin against insulin action in intact rats. In an attempt to elucidate the mechanism of the calcium raising effect of insulin ⁴⁵Ca administered intravenously was used to indicate the movement of calcium from the plasma pool. Insulin administration delayed the plasma ⁴⁵Ca disappearance rate but had no effect on bone ⁴⁵Ca uptake within 120min. In contrast, insulin administration resulted in a 31% reduction of urinary ⁴⁵Ca excretion while the urine volume remained unchanged. However, the insulininduced reduction of urinary calcium excretion could not totally accunt for the calcium raising effect of insulin in TPTX animals.

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A bioassay for thyroid stimulating immunoglobulins (TSI) of patients with Graves' disease was developed by porcine thyroid monolayer cells. Thyroid cells were prepared by dispersion using collagenase and trypsin. Aliquots of the cell suspension (2×10⁶ cells/1.5ml/dish) in Ham's F-12 medium (pH 7.2) containing 10% calf serum and 1.5mM Hepes were seeded and cultured in air at 36 C. On day 6 of culture, cells were incubated with test samples (IgG or bTSH) in 1ml of serum-free, 0.5mM IMX-included fresh medium for an additional time, and cAMP in the cells was measured by radioimmunoassay.
Intracellular cAMP was increased within 5 minutes after the addition of bTSH and the maximal increase was observed after 30min. Responses of cAMP were in a dose-related manner up to 10mU/m1 of bTSH. With the addition of IgG from untreated Graves' patients, dose-related increases in cAMP were also observed up to 10mg/ml IgG and the maximal response was seen at 2 hours incubation. Thyroid stimulating activity in IgG's from normal subjects and patients with Graves' disease was tested with a dose of 10mg/ml and 2 hours incubation and the activity was expressed as a percent of the control (incubated in the same experiment without IgG). One hundred forty one of 145 untreated patients showed higher activity (228±51.8%, mean±SD; 127-393%, range) than normal subjects (103±13.3%, mean±SD, n=24;80-129%, range). Sequential changes in TSI activity in 27 patients after initiating thionamide drugs were studied for 24 months. Initially all 27 patients showed positive TSI and 6 months later 15 remained positive. At 6 months after that, 10 of 23, 4 of 16, and 2 of 6 followed patients showed positive TSI.
These results indicate that this bioassay is clinically useful for detecting TSI.

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We investigated changes in the hypothalamic-pituitary-thyroid axis before, during, and after fasting in twenty-one non-obese euthyroid patients with psychosomatic diseases. Blood samples for free T3 (FT3), T3, free T4 (FT4), T4, reverse T3 (rT3), and TSH were obtained from all patients before and on the 5th day of fasting, and in 11 of the same individuals on the 5th day of refeeding. Serum TSH and T3 responses to TRH were also evaluated in 10 patients before and on the 5th day of fasting. During the fast, FT3, T3 and TSH levels decreased significantly and rT3 levels increased significantly whereas FT4 and T4 levels remained within the normal range. Maximal 4TSH, peak TSH levels, max ΔT3, peak T3 levels, and net secretory responses to TRH decreased significantly. Peak TSH levels and max ΔTSH to TRH correlated well with basal levels of TSH. A statistically significant negativecorrelation between basal levels of FT4 and TSH was observed. After refeeding, there was a significant increase only in TSH which returned to prefasting values. These results demonstrated that in a state of “low T3” during acute starvation a reduction in serum T3 might depend partly on TSH-mediated thyroidal secretion.

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¹³¹I-meta-iodobenzylguanidine (¹³¹I-MIBG) was also taken up by medullary thyroid carcinoma (MTC) as well as by pheochromocytoma in two patients with Sipple's syndrome. However, the mechanism of ¹³¹I-MIBG uptake by MTC has not been clarified yet. We measured tissue catecholamine levels in three MTC, since MTC can produce several active substances. Catecholamines were detected in various amounts in all MTC, but not in normal thyroid tissues. These finding suggest that MTC can produce catecholamines and therefore, ¹³¹I-MIBG is taken up and stored in catecholamine vesicles of MTC, like pheochromocytoma and neuroblastoma. We conclude that ¹³¹I-MIBG may be applied not only to diagnosis but also for the treatment of patients with MTC.

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Effect of aldosterone on the dome formation in the reconstructed MDCK cell epithelia was studied. MDCK cells derived from dog kidney are assumed to be originated from distal tubules or collecting ducts. When cultured to a confluency, these cells formed a epithelial layer with many domes which contained fluid transported from the apical to the basolateral surface through this layer. Aldosterone at a concentration of 10^-8 to 10^-6 M increased the number of domes dose-dependently, probably through a receptor mediated process, since the dome formation induced by this hormone was completely abolished in the presence of spironolactone. This study primarily disclosed that the dome formation in MDCK cells was stimulated by aldosterone, probably through a receptor mediated mechanism.

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The effects of dibutyryladenosine 3', 5'-monophosphate (DBcAMP), a nucleotide analogue, on blood pressure, serum electrolytes and plasma corticoid concentrations were investigated in 10 normotensive healthy subjects who received a constant diet containing 5-8g sodium chloride in hospital. The systolic blood pressure did not change after infusion of 0.25 or 0.33mg/kg/min of DBcAMP for 20min. On the other hand, the diastolic blood pressure was significantly decreased after the infusion of DBcAMP.
The levels of serum sodium and potassium were significantly decreased after the infusion of DBcAMP.
After infusion of 0.25mg/kg/min of DBcAMP for 20min, the changes in plasma levels of 6 corticoids (progesterone, deoxycorticosterone (DOC), 18-hydroxy-deoxycor ticosterone (18-OH-DOC), corticosterone, cortisol and dehydroepiandrosterone sulfate (DHEA-S)) revealed no significant changes.
After infusion of 0.33mg/kg/min of DBcAMP for 20min, the plasma levels of cortisol, corticosterone and 18-OH-DOC were significantly increased and the changes in plasma levels of aldosterone showed a tendency to increase but this was not significant. The plasma levels of DOC and DHEA-S were not appreciably changed, while the plasma levels of progesterone were significantly decreased after the infusin of 0.33mg/kg/min of DBcAMP.
It is speculated therefore that DBcAMP may act to enhance the activity of the sodium-potassium pump and to promote steroid biosynthesis dose-dependently in humans.

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Binding of immunoglobulin G (IgG) to Triton-solubilized fat cell membranes crosslinked with ¹²⁵I-TSH was studied by an indirect immunoprecipitation method. Guinea pig fat cell membranes (FCM) containing TSH receptors with an association constant of 1.92×10⁹M^-1 were first reacted with ¹²⁵I -TSH, then treated with a crosslinker, dissuccinimidyl suberate. The dissociation of ¹²⁵I-TSH from the crosslinked 25I-TSH-FCM complexes due to the addition of 100 mU/ml unlabeled TSH was 9.0%, while it was 33% without the treatment.
To the Triton-solubilized FCM crosslinked with ¹²⁵I-TSH, 50μg each of IgG from 20 normal controls, 20 patients with Graves' disease and 20 with Hashimoto's disease was added and precipitation was effected by adding antihuman IgG. In patients with Graves' disease, ¹²⁵I-TSH-FCM complexes immunoprecipitated ranged from 1.10 to 4.18% with an average of 2.4±0.99 (S. D.)% which was significantly higher than those in normal controls (1.6±0.29%). The values in the patients with Hashimoto's disease averaged 1.7±0.53 (S. D.) which did not differ significantly from those of controls. The value did not correlate with either TSH-binding inhibiting activities or titers of anti-microsomal antibodies. These data suggest the presence of TSHreceptor antibodies which react with antigens other than TSH-binding sites in the patients with Graves' disease.

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To clarify the mechanism for the impaired mineral metabolism in Cushing's syndrome, the clinical features, biochemical parameters before and after oral calcium load, and vitamin D metabolism were compared between two groups of patients of endogenous Cushing's syndrome (17 cases) with and without osteopenia. The patients with osteopenia [OP (+): 7 cases, all female] were older (42.7±8.3 y. o.) and had a longer duration (117±75 M) of the syndrome than those without osteopenia [OP (-): 33.8±8.9 y. o., 36±25M]. OP (-) showed a blunted hypercalciuria after oral calcium load (63.7±20.4 to 90.9±36.1mg/g.Cr), while OP (+) had higher levels of urinary excretion of calcium (fasting: 120.4±37.5, and after oral calcium load: 235.6± 72.6mg/g.Cr), of cyclic AMP (7.6±1.l nmo1/dl·GF), and of plasma 125 (OH) ₂D (76.6±34.0pg/ml) than OP (-)(u-cAMP: 3.2±2.0nmol/dl·GF, 1, 25 (OH) ₂D: 279±16.3pg/ml). These results indicate that 1) elderly fbmale patients with Cushing's syndrome of long duration are susceptible to OP, 2) during the early phases of the syndrome, reduced intestinal calcium absorption with sustained calciuria (probably through the inhibition of calcium reabsorptive effect of PTH by glucocorticoid) induces negative calcium balance, leading to 3) a development of secondary hyperparathyroidism which stimulates 1, 25 (OH) ₂D synthesis. Thus, the mechanism involving bone resorption stimulated by excess PTH along with the direct inhibition of bone formation by glucocorticoid seems to play an important role in a progressive development of OP in Cushing's syndrome.

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Three neonates born to three mothers with primary myxedema who have thyrotropin-binding inhibitor immunoglobulin (TBII) were continually examined after birth. One neonate showed a high TSH level in mass-screening for congenital hypothyroidism and developed transient hypothyroidism. Her TBII disappeared at 114 days of age, and she remained euthyroid after discontinuation of thyroxin replacement at 146 days of age. The other two neonates were euthyroid, though they had positive TBII.
In three mothers, the doses of IgGs that inhibited ¹²⁵I-TSH binding to the level of 50% were compared. The potency of IgG from the mother whose neonate developed hypothyroidism was stronger than that of IgG from the other two mothers. And the elevation of cAMP induced by bovine TSH in suspension culture with porcine thyroid follicles was significantly reduced in the presence of IgG from the three mothers when compared with normal IgG. The thyroid-stimulation blocking activity was more potent in the mother whose neonate developed hypothyroidism than in the other two mothers. This study suggests that the thyroid function of neonates born to primary myxedema with blocking type TBII is influenced by the potency of TSH-binding inhibitor and thyroid-stimulation blocking activity of the mother.

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Placental 20α-hydroxysteroid dehydrogenase (20α-HSD) activity was studied order to evaluate the mechanism of continuation of pregnancy and initiation labor. The placentas obtained at various gestational weeks were homogenized and fractionated into “nuclear”, “mitochondrial”, “microsomal” and “supernatant” fractions. Each fraction was incubated with ¹⁴C-progesterone a hydrogen donor. Enzymatic activity was measured by the conversion progesterone to 20α-dihydroprogesterone. The highest activity of 20α-HSD progesterone was found to be localized in “microsomal” fraction. The Km, constant of 20α-HSD was 4.5×10^-6M for progesterone in “microsomal” fraction. It was found that placental microsomal 20α-HSD required NADPH as well as NADH. 20α-HSD activity for progesterone increased as gestational weeks advanced. The addition of DHA-sulfate and DHA inhibited 20α-HSD activity for progesterone significantly, suggesting that the steroid produced by feto-placental unit may be involved in the metabolism of progesterone in human placenta.

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The effect of α-human atrial natriuretic polypeptide (α-hANP) on the synthesis of prostaglandins was studied in isolated rat glomeruli. Glomeruli were isolated by a passive sieving technique according to the method of Misra and were incubated at 37°C for 60min in the presence (Group II: 10^-6 M, Group III: 10^-5M) or absense (Group I: control) of α-hANP. Furthermore, glomeruli were incubated with arachidonic acid (10^-5, 10^-4, and 10^-3M) and at the end of the incubation period trypan blue was added to the glomerular suspension.
The presence of α-hANP (10^-6 and 10^-5M) caused no significant difference in prostaglandin synthesis as compared with the control. On the other hand, arachidonic acid stimulated prostaglandin synthesis and the glomerular preparation was not stained by trypan blue, indicating that they remained viable. These results suggest that α-h ANP does not directly affect the prostaglandin synthesis in isolated rat glomeruli.

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TSH receptor antibody (TRAb) activity using chicken thyroid receptor (c-TRAb) and porcine thyroid receptor (p-TRAb) was determined by the incubation of 125I-bovine TSH with each receptor. Both c-TRAb and p-TRAb activity in LATS positive and negative Graves' sera were compared. 15 out of 39 LATS positive sera and 4 out of 46 LATS negative sera had positive c-TRAb activity. On the other hand, all LATS positive sera and 33 out of 46 LATS negative sera had positive p-TRAb activity. No relationship between c-TRAb and p-TRAb activity was observed, and there was also no correlation between c-TRAb and LATS activity.
Changes in c-TRAb, p-TRAb and LATS activity in the clinical course of patients with Graves' disease were examined. These activites were parallel in some cases, but in others they were not.
A weak c-TRAb activity was observed in 4 out of 29 Hashimoto's disease, but all cases with thyroid cancer and subacute thyroiditis showed no activity. Sera with positive c-TRAb activity did not stimulate chicken thyroid in chick bioassay. These results suggest that some cases of TRAb in Graves' disease (mainly LATS) inhibit TSH binding to chicken thyroid receptor (non-mammalian species) in the same way as mammalian thyroid, but may not have any stimulatory action on thyroid hormone synthesis. It is interesting to note that TRAb including LATS have the similar effect on TSH receptor even in nonmammalian species.

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The expression of activation antigens (transferrin receptor, IL-2 receptor and Ia antigen) on circulating T lymphocytes from Japanese children with Type 1 diabetes was studied using five monoclonal antibodies (Ab), OKT9, anti-Tac Ab, OKIa1, anti-human HLA-DR Ab and OKT3. For detecting Ia positive T cells, the dual staining technique using OKT3 and anti-Ia antibody was employed. Four out of six patients (67%) with newly diagnosed Type 1 diabetes showed a raised level of either OKT9 or Tac positive cells when examined at diagnosis, These patients, however, rapidly lost these activation antigens after the insulin therapy was started. In contrast, in 32 long-standing patients, only 2 (6%) had a high percentage of OKT9 positive cells and none them demonstrated Tac positive cells. One out of six newly diagnosed patients or three out of 21 long-standing patients had a significantly highpercentage of Ia-positive T cells compared with normal subjects. In poorly controlled long-standing patients whose HbAl value was higher than 14%, none of them had an increased number of activated lymphocytes. Therefore, is unlikely that insulin deficiency and hyperglycemia were responsible for the changes observed in these studies. Activated lymphocytes might be related to activation of the immune system involved in pathogenesis of Type 1 diabetes.

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From three patients with pretibial myxedema (PTM) of Graves' disease, a portion of the skin involved was biopsied, analyzed for proteoglycans and the results were compared with those obtained with euthyroid and hyperthyroid subjects without PTM. The tissue specimen was extracted with 4 M guanidine HCl and subjected to subsequent CsCl density gradient centrifugation. Glycosaminoglycan and protein were recovered in the heaviest density fraction in the three specimens obtained from patients with PTM and not from subjects without PTM. From the analysis by Sepharose CL-6B column, glycosaminoglycan was present as a form of proteoglycan because alkaline borohydride treatment released single chain glycosaminoglycan with a molecular weight of 77, 000 or 66, 000. The digestion with chondroitin ABC lyase revealed that the majority of proteoglycan in the skin tissue was chondroitin sulfate or dermatan sulfate, and heparan sulfate comprised the minor component (14-34%).
The rate of proteoglycan biosynthesis was examined by ³⁵S incorporation into glycosaminoglycan's by cultured fibroblasts from PTM and normal skin. Incorporation of ³⁵S into both proteoglycan and single chain glycosaminoglycan was observed in the fibroblasts of PTM patients as well as of those of subjects without PTM, although the rate of synthesis was more pronounced in the former. The rate of synthesis was influenced neither by normal serum or serum from a pretibial myxedema patient. Since proteoglycan accumulation was detected only in the affected skin of PTM patients, the impairment of local degradation and the proteoglycan clearance mechanism may also be involved.

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Plasma melatonin levels were determined every 20 and 30min for 24 hours on the last day of repeated oral administrations (1 or 2mg a day for 8 or 9 days) of a benzodiazepine derivative (450191-s), which is known to be metabolized to active benzodiazepines after administration. In one of the two subjects, the nocturnal enhancement of plasma melatonin which was obvious on a control day with placebo was diminished almost completely. In the other subject, observed were not only the diminishment of its nocturnal enhancement but also its increase during the daytime almost to the nocturnal levels on a control day, which may indicate a rebound increase in melatonin synthesis or a shift in its day-night rhythmicity.
Such suppressing effects of benzodiazepines on the nocturnal plasma melatonin levels were also examined in the case of a single administration of 2mg of 450191-s or flunitrazepam in the second series of experiments. Even a single flunitrazepam seemed to have lowered nocturnal plasma melatonin levels, which then recovered to the usual levels following the administration of 5mg of a benzodiazepine antagonist, Ro 15-1788, given 6 hours after the flunitrazepam. However, single 450191-s did not show any remarkable effects.
Thus, it has been suggested that benzodiazepines could suppress the nocturnal levels of plasma melatonin or shift its day-night rhythmicity at least when administered repeatedly. The possible action site of benzodiazepines may be the central nervous system, since melatonin synthesis has been thought to be under strongly regulated by the central nervous pathway from the retina to the pineal body. Therefore, these effects of benzodiazepines may provide a method for investigating the physiological role of melatonin and its day-night rhythmicity as well as to further clarify the system regulating melatonin synthesis in humans.

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We have developed and evaluated a new and simplified method for the detection of thyroid hormone autoantibodies (THAA) in serum. The method includes acidification of serum followed by adsorption of liberated thyroid hormones onto dextran-coated charcoal and then alkalinisation of the serum in assay buffer prior to performing a binding study. Using our method, specific binding of ¹²⁵I-T₄ to serum THAA in two patients with Hashimoto's thyroiditis was almost the same regardless of whether or not the sera had been preincubated with a large amount of cold T₄. On the other hand, without the acid treatment, preincubation with cold T₄ considerably inhibited the binding of ¹²⁵I-T⁴ to serum THAA in both cases. These results indicate that serum THAA can be easily detected under conditions in which circulating thyroid hormones hardly affect the binding study by using our new sensitive method.

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RU 27987 is a new ligand for progesterone receptor and binds in high affinity to nuclei of target tissues of progesterone. Using this compound, progestin-binding components in the benign hypertrophic human prostate were studied, and compared with those examined with R 5020, a conventional ligand, in the study of progesterone receptor. In cytosols, the binding affinity of RU 27987 was higher than that of R 5020, and the number of maximum binding sites for RU 27987 seemed to be large but correlated well with those of R 5020. The binder for RU 27987 sedimented at 8.6 S, and the binding was specific to progestational steroids, indicating that binding properties of this binder in the cytosols are identical to those for R 5020.
Although there was no binding with R 5020 in the nuclear extract, a small amount of specific binding with RU 27987 was detected. However, the cytosol bound with RU 27987 was not retained in DNA Sepharose and no specific binder for RU 27987 in the nuclear extract was observed in a sucrose density gradient centrifugation. From these observations, it was assumed that the nuclear binding observed was attributable to contamination of the cytosolic binder. The results obtained in the present study suggest that the progestinbinding component in the benign prostatic hypertrophy is not the progesterone receptor but a high affinity binder for progestins whose physiological role is not clear at present.

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