Plasma GH levels basally and after CDP-Choline (300mg) i. v. administration were evaluated by radioimmunoassay in patients with brain lesions variously located (frontal lobe, temporal lobe or hypothalamus) as well asinsubjects with pituitary lesions. The results were statistically analyzed and compared with those obtained in normal subjects. A lack of response to CDP-Choline was observedin patients with pituitary adenomas or hypothalamic lesions. The GH response was delayed in patients with frontal lobe diseases. On the basis of the results obtained, we discuss the role of the different central nervous structures on the control of the GH activating system.

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The effects of calcitonin (CT), epinephrine and glucagon on the plasma membrane Ca-ATPase activity and the calcium content in the liver were investigated 30min after a single subcutaneous administration of hormones to rats. Ca-ATPase activity in the plasma membrane fraction was significantly decreased by CT (80 MRCmU/100g BW), while it was not significantly lowered by insulin (100mU/100g BW), epinephrine (100μg/100g BW), glucagon (50μg/100g BW), or parathyroid hormone (25U/100g BW). The calcium content in the liver was markedly increased by CT, while it was not significantly elevated by epinephrine or glucagon. Meanwhile, the decrease of Ca-ATPase activity in the plasma membrane fraction produced by CT was significantly prevented by simultaneous administration of epinephrine or glucagon, and also the increase in liver calcium was noticeably interfered with. The present results suggests that the action of CT on liver calcium may differ from that of epinephrine or glucagon which causes an increase in cyclic AMP in the liver cells.

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The properties of 5α-reductase in the human epididymis were studied. When testosterone-4-¹⁴C was incubated with the whole homogenate of human epididymis in the presence of NADPH, the identified metabolites of the substrate were 5αdihydrotestosterone and 5α-androstane-3a, 17β-diol. 5α-reductase activity was mainly localized in the microsomal and nuclear fractions. The Km values of the 5α-reductase for testosterone in the microsomal and nuclear fractions were 1.07×10^-8M and 0.83×10^-8M, respectively. The optimum pH of the 5α-reductase activity was 5.7 for the microsomal fraction and 5.5 for the nuclear fraction. The optimum temperature was about 48°C for 5α-reductase in both the microsomal and nuclear fractions. The most preferable cofactor for the microsomal 5α-reductase was NADPH and the Km value of the enzyme for NADPH was 2.1×10^-6M.
These results confirm the presence of 5α-reductase in the human epididymis and suggest that the enzyme has considerable affinity for testosterone and that the properties of 5α-reductase in the microsomal fraction are similar to those in the nuclear fraction.

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Serum LH and FSH levels were determined before and after LH-RH injection (100μgi.m.) in patients with prostatic cancer who were chronically treated with either chlormadinone acetate (CMA, 100 mg/day) or ethynylestradiol (EE, 1 mg/day). In patients treated with EE, the levels of serum LH and FSH before and after injection of LH-RH were significantly lower than those in controls. On the other hand in patients treated with CMA, the basal levels of serum gonadotropins did not differ from those in controls, and the increase in gonadotropin after LH-RH injection was comparable to that in controls.
To examine the effects of these steroids on the hypothalamo-hypophysial axis in the regulation of gonadotropin secretion, CMA or EE was implanted in castrated male rats. CMA, EE or cholesterol (control) was implanted in the hypothalamic median eminence-arcuate nucleus region through a stainless doublecannula. EE implantation resulted in a 75% decrease in serum LH (p<0.001) and a 38% decrease in serum FSH (p<0.05) from the control levels on day 5 of implantation. On the other hand, CMA implantation induced a 33% decrease in serum LH (p<0.05) from the control level on day 3 of implantation, but no significant change in serum FSH levels. The injection of 2 μg/kg of LH-RH on day 7 of implantation induced significant lowering of LH and FSH levels. There was no significant difference between serum levels of the hormones 20 min after LH-RH injection for these two groups and those for the control group.
These studies suggest that EE has a potent negative feedback effect on both LH and FSH secretion, and that CMA has a mild negative feedback effect on LH secretion.

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In order to investigate the effect of 3, 5, 3'L-triiodothyronine (T₃) administration on thyroid hormone concentrations in serum, thyroxine (T₄), T₃, 3, 3', 5'L-triiodothyronine (reverse T₃, rT₃) and thyroid stimulating hormone (TSH) concentrations in serum were determined before and after T₃ administration in 10 hypothyroid patients maintained on constant doses of T₄.
Ten hypothyroid patients given 100 μg of T₄ for approximately 3 months had almost normal T₄ and T₃ concentrations in serum. Seven patients showed almost normal rT₃ concentrations in serum and they were slightly diminished in the remaining 3 patients. TSH levels in serum were almost within the normal limit in 7 out of 10 patients. However, despite the elevation of T₄ and T₃ levels, 3 patients had markedly elevated TSH levels.
Values for serum T₄ concentrations were decreased 4 weeks after the administration of 50 μg T₃ in all patients maintained on constant doses of T₄, although they were almost within the normal range. T₃ concentrations in serum, which was obtained just before the administration of the next daily doses of T₃, were markedly elevated in 6 of 10 patients after T₃ administration and the remaining 4 patients had also slightly higher T₃ concentrations than those before T₃ administration. On the other hand, serum T₃ concentrations were diminished in 5 patients during T₃ ingestion. They were somewhat diminished or almost unchanged before and after T₃ administration in the remaining 5 patients. Moreover, 3 patients with elevated TSH levels during T₄ administration showed almost normal TSH levels after T₄ and T₃ ingestion.
The results showed the reciprocal relationship between T₃ and rT₃ levels in serum after T₃ administration in hypothyroid patients maintained on constant doses of T₄. Furthermore, the present findings suggest that the administration of both T₄ and T₃ might be a more suitable replacement therapy in the patients with hypothyroidism than T₄ alone.

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Anterior (AHD) and complete hypothalamic deafferentation (CHD) were performed in female rats to ascertain the origin of LHRH detected in the external layer of ME. Deafferented brains were serially sectioned in a cryostat in the frontal plane. LHRH activity in each section was determined by RIA. While AHD, using a knife with a small radius caused no change in the distribution of LHRH in the brain, AHD, using a knife with a large radius resulted in a significant decrease in LHRH content in the ARC-ME. CHD caused a more marked decrease in the ARC-ME LHRH than AHD. The decrease was more marked in the anterior and posterior portions of the ARC-ME than in the middle one. These effects of CHD on the distribution of LHRH in the ME were ascertained by immunohistochemical studies. In contrast, LHRH content in the POA showed no significant change after AHD and CHD. However, a few LHRH containing fibers were observed in the area lying just proximal to the cut by immunohistochemistry. These results strongly suggest that a part of the LHRH detected in the external layer of ME is derived from that synthesized outside the MBH, possibly in the POA and another part is derived from that synthesized inside the MBH, possibly in the ARC.

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Effects of estradiol hemisuccinate and diethylstilbestrol diphosphate on the cardiovascular system in male rats were examined in vivo and in vivo. In conscious rats, intravenously administered estradiol or diethylstilbestrol inhibited vascular responses to norepinephrine in a dose-dependet manner. Diethylstilbestrol in high doses significantly lowered the blood pressure of conscious rats.In the isolated mesenteric arteries perfused with Krebs bicarbonate solution, estradiol or diethylstilbestrol in the perfusate similarly inhibited vascular reactivity to norepinephrine in a dosedependent manner. As in the in vivo experiments, adecrease in the basal pressure of the mesenteric vascular bed was observed when diethylstilbestrol was added to the perfusate at high concentrations. The results of these in vivo and in vitro studies strongly suggest that estradiol and diethylstilbestrol act directly on the vascular beds and attenuate vascular response to norepinephrine. It is also suggested that diethylstilbestrol by itself causes vasodilatation and areduction in blood pressure when present at high concentrations.

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The existence of testosterone-binding component in the cytosol fraction of the male rat liver was shown. The binding ability of the component with testosterone was decreased by the addition of excess unlabeled steroids, but the steroid binding activity of the serum was not affected. The Scatchard plot indicated that the component has a moderate binding affinity with testosterone and the number of the binding sites of the component is limited.

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A study was made of the nature of a macromolecular component in the hepatic cytosol of male rats which binds with testosterone and androstenedione. The saturation analysis revealed that this component has binding affinity with these androgenic steroids and a limited number of binding sites. This component was inactivated by incubation with proteolytic enzymes and by heating, but was unaffected following incubation with RNase. Analysis by sucrose density gradient centrifugation revealed that this component had a sedimentation coefficient of approximately 10S. On the basis of the elution profile of the component on gel chromatography, the molecular weight and the similarity of the structure of these androgens, it seems most likely that one binding component is responsible for both testosterone and androstenedione. It was postulated that the only difference in binding for these androgens is that testosterone showed a more rapid association rate with this component, than androstenedione.

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In order to examine the possibility that the changes in electrolytes in tissue alter the effect of thyroid hormone on NaK-ATPase, rats were fed either synthetic K-deficient diet or synthetic K-normal diet. K-deficient diet induced a reduction in K content in serum or kidney, while that of the liver remained unchanged. When adaily dose of 2.5μg T₃ was administered for 7 days to k-deficient rats, both Mgand NaK-ATPase of the homogenate of liver and kidney were elevated, while the same dose failed to influence those enzymes in K-normal rats. Furthermore, T₃ dose increased the Na content of liver and kidney in K-deficient rats, resulting in a significant decrease in the K/Na ratio in those tissue. Based on the estimation from chloride space, the decrease in K/Na was deemed to have occurred mainly in the intracellular space. As the levels of serum thyroid hormone and liver T³ were not influenced by Kdeficiency, the effect of K depletion is likely to be mediated not through the alteration in thyroid hormone kinetics, but through some other mechanism such as the elevation of intracellular Na. The present study demonstrates that K deficiency may sensitize NaK-ATPase to the effect of thyroid hormone.

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A modified erythrocyte insulin receptor assay was developed. The major advantage of our method is the smaller amount of blood necessary for the assay and consequent simple handling in the binding assay. Three ml of blood is enough for this assay, making it possible to assess insulin receptors in a group of pediatric patientts. There was a close relationship between mononuclear cells and erythrocyte insulin binding assays (r=0.900, p<0.05).
With this method insulin binding studies were done on normal subjects and adult onset non-obese diabetics. Decreased insulin binding was seen in the diabetic group, and this was due to a decreased number of insulin receptors.
This erythrocyte insulin receptor assay can be a useful tool for the study of the mechanism of insulin resistance.

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In order to ascertain whether insulin secretion is inhibited by insulin per se, the effect of exogenous rat insulin on basal and stimulatedrat C-peptide reactivity (CPR) release was studied in the isolated perfused rat pancreas. The pancreas was perfused with a medium containing 20 or 200ng/ml of rat insulinfor a 20 min equilibration period and successively for a 25 to 30 min test period during which time glucose (300mg/100ml), tolbutamide (500μg/ml), glucagon (500ng/ml) or arginine (10mm) was added as a secretagogue. The concentration of glucose in the basal medium was 60mg/100ml. Exogenous insulin did not significantly suppress the basal CPR secretion, nor did it cause a suppression of the peakvalue or incremental area of CPR while the pancreas was stimulated, but it rather augmented them. No inhibitory effect of exogenous insulin on the basal or stimulated CPR release was noticed in the present study.

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PTH (0.5 unit/ml) and CT (0.5 unit/ml) strongly stimulated the in vitro growth of the chick embryo femur in terms of elongation and dry weight increase as well as an increase in the protein, hexosamine, hydroxyproline, DNA and RNA content of the femur. A synergistic effect was observed when PTH and CT were simultaneously added to the medium, suggesting that the mechanism of the growth stimulating effect of one of these hormones might be different from that of the other. The exposure of PTH to the femur caused an increase in cyclic AMP content. The direct addition of dibutyryl cyclic AMP (1 mm) to the medium mimicked the effect of PTH on the cartilage. PTH, therefore, seems to stimulate the growth of cartilage via the increase in cyclic AMP content of the femur. CT had no effect on the cyclic AMP content of the femur.

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We have found that the developmental stage of chondrocytes in the chick embryo femur is sensitive to the action of PTH, CT and dibutyryl cyclic AMP in stimulating proteoglycan synthesis as estimated by the incorporation of radioactive inorganic sulfate. PTH treatment of the cartilage resulted in dramatic stimulation of the proteoglycan synthesis in the diaphysis, which is composed of rather old non-dividing hypertrophied chondrocytes with much lower proteoglycan-synthesizing activity than younger dividing cells in the epiphysis and metaphysis. No or slight stimulation was observed the epiphysis and metaphysis. Dibutyryl cyclic AMP produced much the same effect. CT also stimulated the proteoglycan synthesis only in the diaphysis but the stimulation was less potent (133% of control) than PTH (650% of control) or dibutyryl cyclic AMP (625% of control). The hormones and dibutyryl cyclic AMP, therefore, seem to activate the proteoglycan synthesis specifically in rather old chondrocytes of the diaphysis. A synergistic effect of PTH and CT suggesting a different stimulating mechanism was observed in the diaphysis.

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In an attempt to clarify the mechanism responsible for the prolonged effect of DDAVP (1-desamino-8-D-arginine vasopressin), plasma levels of DDAVP and nephrogenous cyclic AMP were determined in patients with diabetes insipidus after a single intranasal administration of 10μg of DDAVP. Plasma DDAVP levels were uniformly elevated within 30 min, and showed a peak ranging from 5.6 to 25.0pg/ml between 30 and 120 min. The subsequent time-course of plasma DDAVP differed however, from patient to patient, and was irregular in most of them. In all of the patients whose plasma DDAVP dropped below 1.0pg/ml, antidiuresis was still observed. Although the mean basal level of nephrogenous cyclic AMP in patients with diabetes insipidus was not significantly different from that in control subjects, the administration of DDAVP resulted in a 2-fold increase. A negative correlation between nephrogenous cyclic AMP and free water clearance was obtained. These results suggest that the longacting nature of DDAVP may be attributed, in addition to its gradual absorption from nasal mucosa and slow metabolic clearance, to a higher or persistent biological activity at the receptor site in the kidney and that a nearly physiological level of antidiuretic hormone may cause de novo synthesis of cyclic AMP in the kidney and exert its biological action.

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Radioimmunoassayable TRH and TSH were measured in plasma samples taken at 5 min intervals for 4 hr (2100-0200 hr) from 4 normal male subjects. Three subjects showed a TSH surge at 2135 hr, 2455 hr and 0150 hr, respectively. The mean plasma TRH level of the 4 subjects was 10.3-11.7pg/ml. Plasma TRH showed random fluctuation, which did not coincide with the nocturnal increase in plasma TSH.

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A double antibody heterologous enzyme immunoassay was developed for measuring thyroxine (T₄) in serum and in dried blood samples on filter paper for use in screening for neonatal hypothyroidism. In this method, ethanol extracts of 100 μl of serum or dried blood spots of 9 mm in diameter were incubated with Tralkaline phosphatase conjugate prepared with glutaraldehyde, and antiserum to T₄-bovine serum albumin conjugate prepared with carbodiimide. The enzyme activity in the precipitates formed with second antibody was then determined photometricallty at 500 nm. The measurable range of T₄ was 1-32μg/dl. A linear relation was obtained between the sample volume and the T₄ value, and a high correlation was found between values for T₄ determined by this method and those for T₄ in serum determined by radioimmunoassay (coefficient of correlation: r=0.97 for serum and r= 0.88 for dried blood samples).

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The effect of calcium chloride (4.0mg Ca²⁺/100g BW) on the calcium metabolism in the hepatic bile system was investigated in intact and thyroparathyroidectomized rats. The administration of calcium did not alter the serum calcium of intact rats but increased it markedly in thyroparathyroidectomized rats. The administration of calcium to intact rats also caused a significant decrease in the liver plasma membrane Ca-ATPase activity, and a corresponding increase in liver calcium and bile calcium, while no such increase was seen in thyroparathyroidectomized rats. Meanwhile, the administration of calcitonin (CT, 80 MRC mU/100g BW) to intact rats significantly lowered both serum calcium and liver plasma membrane Ca-ATPase activity, and it also significantly elevated both liver and bile calcium. These results suggest that endogenous CT regulates calcium metabolism in the hepatic bile system of rats after the injection of calcium chloride.

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Morphological and biochemical studies were performed in pancreatic islet cell tumors found in rats given alloxan (40mg/kg) and nicotinamide (305mg/kg). Complete serial sections of the whole pancreas, combined with planimetric analysis, uncovered islet cell tumors in 5 of 7 rats which were killed 10 to 14 months after treatment. Hypoglycemia associated with hyperinsulinemia was found in a rat which developed a tumor which consisted of cells which reacted lightly with aldehyde fuchsin. Another rat developed a gross tumor which was composed of cells stained deeply with aldehyde fuchsin. However, flat insulin response to glucose associated with glucose intolerance was found in this rat. In addition to B cells, a few A and D cells were found within the two tumors.
The present study suggests that pancreatic islet cell tumors found in rats given alloxan and nicotinamide are composed of at least three endocrine cell populaticns, although the majority of tumor cells are insulin-producing B cells.

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Characterization of estrogen-binding components was attempted in cytosol fractions from thymus, spleen and mesenteric lymph node of castrated rats. As shown by sucrose gradient analysis, specific binding of [6, 7-³H] estradio1-17β in the thymus is associated with a component migrating at 4 S. The binding of [6, 7-³H] estradio1-17β is highly specific since it is easily displaced by unlabeled estradio1-17β and diethylstilbesterol. Affinity of unlabeled estrone, estriol and clomiphene citrate, is much lower, and estradiol-17α, progesterone, testosterone, 5α-dihydrotestosterone and corticosterone have no affinity for the component at all. The dissociation constant of thymic estrogen binding is 0.25 nm in males and 0.3 nm in females. The number of binding sites is 6 fmols/mg protein in both sexes. No specific binding to estrogen is, however, found in cytosol fractions from the other two tissues.
Enzyme-and heat-experiments demonstrate that specific estrogen binder in thymic cytosol is heat-labile and at least protein in nature. It is concluded that the rat thymus contains a cytoplasmic estrogen receptor which is in part protein and heatlabile.

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Clofibrate has been considered to be a relatively safe antidiuretic in the treatment of diabetes insipidus. However, we have recently had four cases of clofibrate-induced myopathy in patients with diabetes insipidus due to hypothalamic lesions. Physicians should therefore be aware of its occurrence and carefully monitor serum levels of CPK, GOT and GPT during the treatment of diabetes insipidus with clofibrate, especially in patients with associated hypothyroidism, latent or overt, which possibly favors the development of myopathy.

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