A31-year-old man and a12-year-old girl were diagnosed as pseudohypoparathyroidism (PHP) Type I because of a failure to respond to the administration of parathyroid hormone (PTH) with increased urinary excretion of phosphate and cyclic adenosine-3', 5'-monophosphate (cAMP). A 22-year-old woman was diagnosed as PHP Type II because there was no increase in the urinary excretion of phosphate despite of a marked increase in urinary cAMP excretion. With the combined calcium-PTH infusion or PTH infusion after vitamin D therapy, renal response was improved in these patients. Also dibutyryl adenosine-3'-5'-cyclic monophosphate (dbcAMP) infusion evoked an increased urinary phosphate excretion in all of the patients. The metabolic defect of our patients with PHP Type I may be caused not by a lack or defective form of PTH-sensitive receptor adenylate cyclase complex but rather by an abnormal conformation in the plasma membrane-associated receptor adenylate cyclase enzyme complex in kidney. In the patient with PHP Type II, as cAMP generation is intact, the metabolic defect might be related to a defect of calcium mobilization in renal tubular cells in response to PTH.

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Sucrose density gradient ultracentrifugation and dextran-coated charcoal adsorption permitted us to characterize the estrogen-binding proteins in cytosols obtained from the thymus, spleen and mesenteric lymph node of the castrated male and female mice of C57BL strain. The thymic cytosol from both sexes incubated with 3H-estradio1-17β in the presence of excess unlabeled steroids showed a specific estrogen binding 4S protein with its binding capacity of 10^-14 moles/mg protein for males and 4×10^-15moles/mg protein for females, respectively. The dissociation constant was of 4×10^-10M for males and 3×10^-10M for females, respectively. No specific binding was, however, found in the cytosols of the spleen and mesenteric lymph node.
Steroid analysis by thin-layer chromatography of the thymic cytosols after incubation of them with 3H-estradio1-17β showed that a fair amount (around 60%) of radioactivity was from the undegradated radioactive steroid still bound to4S binder in both sexes. Enzyme study and heat experiment revealed that the estrogen specific 4S binding component in the thymic cytosols bears at least protein in nature and is of heat-labile nature. These results strongly suggest that the thymus of the castrated mice contain a sspecific estrogen receptor, the nature of which is in part protein and heat-labile.

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Our previous study, in which we measured serum17α-hydroxyprogesterone (17-OHP) every 8hr during the periovulatory phase in women, suggested that17-OHP may have a physiological role in inducing and/or facilitating LH release. Therefore, this study was designed to elucidate the possible role of 17-0HP in LH release.
Experiment 1: Three weeks after ovariectomy, rats were injected subcutaneously with 20 fig of estradiol benzoate (EB) at 1200hr (day0). The administration of 2mg of progesterone (P) or 17-OHP was performed systemically at 1200hr on day3. The injection of P elicited a significant increase of serum LH 6hr later as compared to the serum levels in the control group (p<0.05), but the injection of 17-OHP failed to alter serum LH significantly.
Experiment 2: Three weeks after ovariectomy, rats were injected with EB at 1200 hr. Crystalline P or17-OHP was inserted through the outer cannula into the brain at1200hr on day3. All animals bearing17-0HP implants or P implants in the diagonal band of Broca (DBB) exhibited an increase in serum LH6hr after implantation. In regard to implants in the medial preoptic area (MPO), all rats with implants of 17-OHP and 4 of 7 with implants of P exhibited an increase in serum LH, whereas both17-0HP and P implantations in the septum failed to induce an increase in serum LH.
Experiment3: All rats were injected subcutaneously with 20 fig of estradiol (E2) rather than with EB. Vaginal cornification was observed in 100% at 1200hr on day 3 after the injection of EB, but in only5.5% at 1200hr on day 3 after the injections of E2. In rats with implants in the DBB or the MPO, no significant increase of serum LH was observed after implantation of 17-OHP or P.
These results suggest that the sites of stimulatory feedback action of 17-OHP on LH release are similar to those of P and that the stimulatory effects of 17-OHP are synergistic with estrogen in the induction of the LH surge.
Results from the present study, in conjunction with our previous observation, strongly suggest a physiological role for 17-OHP in inducing the midcycle LH surge in women.

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Immunohistochemical localization of hCG and its subunits in chorionic tissue of early gestation was carried out.
Antibodies to purified hCG and its subunits were obtained by using these agents for immunization according to the small doses method. The antibody titers and specificities were examined by B/T and standard curves in homologous radioimmunoassay system.
The tissue preparations were stained both by a direct and by an indirect method utilizing these antisera and observing the specimens under a fluorescent microscope.
The results were as follows. 1) With the anti-hCG staining, fluorescence was observed in the syncytiotrophoblasts as reported previously while the cytotrophoblast were stained slightly.
2) with the anti-hCG-β staining, the fluorescence was almost identical with that of hCG and showed a more distinct pattern.
3) with the anti-hCG-α staining, the fluorescence was found both in the syncytioand cytotrophoblasts concurrently. Fluorescence of the latter cells was recognized as due to free a-subunit because cytotrophoblast was scarcely stained with anti-hCG and anti-hCG-β.

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A genetically diabetic model, KK-CA^Y mice which were bred by mating female KK mice (aa, BB, cc) with male KK-CA^Y mice (A^Ya, BB, CC) was studied on the usefulness as a tool for a pharmacological assay. Body weights of KK-CA^Y mice increased more rapidly than those of control mice, KK-C. When the body weights of male KK-CA^Y mice reached, about 30g 10 weeks after birth, their blood glucose levels increased. Severe hyperglycemia (over 300mg/100ml) was often observed in the males, but not in the females. Glucose tolerance in the KK-CA^Y mice was more markedly impaired than that in the control mice. The increase in blood FFA level correlated with the increase in body weight on both KK-CA^y mice and the controls. On hyperinsulinemia observed, the ratio of plasma immunoreactive insulin (IRI) level to blood glucose level in the male mice was lower than that seen in the female mice. On hyperglucagonemia observed, elevation of plasma immunoreactive glucagon (IRG) was more remarkable in the males than in the females. Morphological study showed insular degranulation only in the males. Since the dose-dependent insulin-induced falling was observed on blood glucose level in nonfasted KK-CA^y mice, they could be used as a feasible tool for an assay of antidiabetic drugs.

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Effects of estrogen treatment (estradiol benzoate 20μg per day for 2days) on plasma concentration of oxytocin and vasopressin were studied in ovariectomized female rats under urethane-anesthesia (1.2g/kg). Plasma oxytocin was measured by bioassay, while plasma vasopressin was determined by both bioassay and radioimmunoassay. Plasma oxytocin levels (46.2-232.2μU /ml) in the jugular blood of estrogentreated rats were found to be significantly (p<0.05) higher than those (non-detectable level to130.0μU/ml) of the ovariectomized control rats. The mean plasma vasopressin levels determined by bioassay were 172±66 (mean±SEM)%mu;U/ml in control rats and 176±32μU/ml in estrogen-treated rats. The mean concentrations of immunoreactive plasma vasopressin were60.1±19.6μU/ml in the control and75.5±26.8μU/ml in estrogen treated rats. Thus no significant change in plasma vasopressin due to the estrogen treatment was found, whatever method was employed to determine its concentrations.
Anterior or posterior hypothalamic deafferentations were made in estrogen-treated ovariectomized rats. The facilitatory effect of estrogen on oxytocin release was not found in the rats with anterior deafferentation. The plasma oxytocin level in the estrogentreated rats with the anterior deafferentation was significantly (p<0.05) lower than that in the estrogen-treated rats without the hypothalamic deafferentation. On the other hand, the plasma oxytocin level in the estrogen-treated rats with posterior deafferentation did not show any significant difference from either that of ovariectomized control rats or that of estrogen-treated ovariectomized rats with intact brain.
The present data indicate that estrogen stimulates oxytocin release in the female rat and that such an action of estrogen is exerted mainly by activating the anterior neural input to the neurosecretory cells in the hypothalamo-neurohypophysial system. Whether or not estrogen gives influence on the posterior neural input to the neurosecretory cells is equivocal in the present experiment.
An additional experiment was made to determine the effect of urethane on plasma vasopressin. Urethane-anesthetized rats showed a significantly (p<0.01) higher plasma vasopressin level than conscious or pentobarbitone-anesthetized rats.

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Human calcitonin (hCT) response to a 1-minute calcium infusion was studied in 6 male and 4 female young normal subjects and in 6 male and 6 female elderly subjects. In the young subjects plasma hCT levels increased significantly (p<0.01) from mean basal value of82.3±48.7pg/ml to maximum level of 407.5±198.0 pg/ml in the male, while it increased from 96.3±89.2 to 216.3±89.1 pg/ml (p<0.05) in the female. In the elderly subjects, it increased from 80.3±56.9 pg/ml to maximum level of 229.2±130.6 pg/ml (p<0.05) in the male, while it increased from 109.2±43.7to 163.3±55.7 pg/m/(p<0.005) in the female.
There was no significant difference in the basal hCT level of these 4 groups. In both the young and the elderly, however, mean increment of hCT level was significantly (p<0.05) higher in the male than in the female of the same-aged group. In both the female and the male subjects, it was significantly (p<0.05) higher in the young than in the elderly of the same sex. A1-minute infusion of calcium can be used as a reliable provocative test for hCT secretion in human subjects.

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The sera for 88 parents and 9 siblings of 73 patients with insulin dependent diabetes mellitus in childhood and 437 controls matched in age and sex, were tested by the thyroglobulin and microsome-coated tanned red cell hemagglutination test (Fuji-Zoki Co. Tokyo).
None of 73 children with diabetes mellitus had antithyroglobulin antibodies, whereas twelve (16.4%) had antimicrosomal antibodies compared with the incidence of 0.4% and 1.1%, respectively, in 437 controls.
In the parents and siblings of these probands, thyroid antibodies were also found in increased incidence.
The incidence of antimicrosomal antibodies in the 68 mothers was significantly higher than in controls matched for age and sex, but the incidence of the positive thyroid antibodies in the 20 fathers and 9 siblings was not significantly different from that in control populations. The incidence of thyroid antibodies tended to be higher, though not significant, in parents and siblings of diabetic children with positive thyroid antibodies than in those of diabetics with negative ones.
These findings suggest that immunogenetic factors may be responsible for the pathogenesis of some cases of diabetes mellitus in childhood.

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Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa (the capsular fraction) from the rat adrenal cortex have been studied. Although high concentrations of extracellular calcium potentiated the stimulatory action of ACTH on cyclic AMP and aldosterone output, tetracaine or verapamil inhibited aldosterone output but not cyclic AMP production during ACTH-stimulation. Lanthanum reduced both aldosterone and cyclic AMP accumulation induced by ACTH. These results suggest that an extracellular calcium would be essential in stimulating the capsular steroidogenesis without involvement of the cyclic AMP system.

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The correlation between corticoidogenesis and Ca²⁺-influx in the cell was investigated using isolated rat or bovine adrenocortical cells.
1) ACTH-induced corticoidogenesis in both rat and bovine adrenocortical cells was enhanced in parallel with increasing concentrations of external Ca²⁺. In the bovine cells but not in the rat cells, moreover, the marked stimulatory effect of external Ca²⁺ on the corticoidogenesis was observed despite the absence of ACTH. 2) Ca²⁺-influx and corticoidogenesis always occurred unitedly. 3) Verapamil markedly inhibited either corticoidogenesis or Ca²⁺-influx in response to ACTH. 4) The stimulatory effect of Ca²⁺ on corticoidogenesis was completely blocked by cycloheximide.
It was therefore suggested that Ca²⁺ could regulate corticoidogenesis as a primary “second messenger” of ACTH through biosynthesis of so-called steroidogenic protein.

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When isolated rat liver cells were incubated in the presence of vasoactive intestinal peptide at the concentrations ranging from 0.2μg to 2μg per ml, glycogenolysis was maximally stimulated within 15min. However, somatostatin inhibited the liver glycogenolysis. The combined addition to the incubation medium showed that insulin and somatostatin inhibited the stimulated glycogenolysis induced by vasoactive intestinal peptide, while vasoactive intestinal peptide plus secretin showed no additive effect on glycogenolysis, as compared with single the addition of vasoactive intestinal peptide. On the other hand, the addition of glucagon to vasoactive intestinal peptide showed additive effects on glycogenolysis. These results suggest that the receptor site for vasoactive intestinal peptide may be distinguishable from that for glucagon. Extracellular calcium ions were demonstrated to play an important role in the modulation of vasoactive intestinal peptide-induced glycogenolysis.
The evidence presented in this paper indicates that glucose metabolism may be partly regulated by the direct action of vasoactive intestinal peptide on hepatocytes, which is referred to as an enterohepatic axis and that the axis is inhibited by insulin and somatostatin.

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The effects of vasopressin and cyclic AMP on water transport at arachnoid villi into the superior sagittal sinus were examined using the isolated meninges preparations of cats. The meninges preparation, the superior sagittal sinus of which was opened at the midline of the outer surface, was held between two polyethylene tubes. The tubes were fixed vertically in the way that the opened surface of the sinus was directed downward and arachnoid surface upward. Water transport was determined by measuring the tritiated water dripping through the membrane preparation. Vasopressin from less than 50 to 500μU/ml accelerated the water transport and this effect was dose-dependent. Cyclic AMP from 0.5 to 10 mM was proved to manifest the same effect as vasopressin. This effect of cyclic AMP appeared rapidly in comparison with that of vasopressin, suggesting that the effect of vasopressin may be manifested through cyclic AMP. From these findings a physiological role of vasopressin in cerebrospinal fluid was discussed regarding the regulation of intracranial pressure.

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Insulin and cyclic adenosine 3', 5'-monophosphate (cAMP) release from the perifused islets was examined under a slow-rise stimulation by D-glucose, D-galactose and the anomers of D-glucose, using a technique of the double chamber method.
The insulin and cAMP secretory responses of the B cell to D-glucose showed sigmoidal curves with K_m of 6.6±1.0mM glucose for insulin release and with K_m of 6.0±0.7mM glucose for cAMP release. The respective K_m values of α-and β-D-glucose were 4.5±0.3 and 5.9±0.2mM for insulin release, and 4.0±0.6 and 5.3±0.9mM for cAMP release. The thresholds for insulin and cAMP release due to the α anomer were 4.0and 3.0mM glucose, respectively, while the respective thresholds of the β anomer were 4.6 and 4.0mM for insulin and cAMP release, the former being more potent than the latter in stimulating insulin and cAMP release. Furthermore, it seems obvious that the release of cAMP slightly precedes insulin release at low concentrations under a slow-rise stimulation with the anomers. These results suggest that cAMP may be released not only from the B granules by exocytosis but also from another compartment located in the cell membrane or near the membrane. Hill plots for the rates of cAMP and insulin release were represented as straight lines but Lineweaver-Burks plots were parabolic functions in all cases.
Consequently, these data suggest that the pancreatic B cell may contain glucoreceptors and the binding of the glucose molecules to the receptors may directly cause the activation of the adenylate cyclase, which, in turn, leads to the accumulation of cAMP in islets and perifusion medium.

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Using B-mode ultrasonography, an attempt was made to measure the volume of extraocular muscles and retrobulbar fat in 31 patients (62 orbits) with Graves'disease. None of the patients had exophthalmometric measurements greater than 21mm or had eye symptoms. The mean value of muscle volume of Graves'patients was significantly larger than that of normal controls (6.48±2.70cm³ and 3.25±1.30cm³, respectively, p<0.001). All of the patients had extraocular muscle swelling, although 2 of them had no extraocular muscle change for their unilateral eye. The extraocular muscle volume increased as the degree of the proptosis increased. The fat volume tended to increase in parallel with the degree of the proptosis. In the Graves' group with obvious proptosis (Hertel reading: 19-21mm), the fat volume increased more significantly than in any other group. The ratio of extraocular muscle volume to retrobulbar fat volume was significantly higher in Graves' disease, but it did not increase as the degree of the proptosis increased. A significant correlation between proptosis and muscle volume plus fat volume was observed. No significant difference of the extraocular muscle volume was observed between the patients untreated and treated with antithyroid drugs. The data show a uniform enlargement of the extraocular muscles in Graves'disease and also suggest an involvement of increased retrobulbar fat volume in a group of obvious exophthalmos. The degree of the proptosis is in aclose proportion to the quantitative change of the orbital soft tissue.

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A modified equilibrium dialysis method to determine the percentage of protein-unbound cortisol in the total serum cortisol (unbound cortisol %) was devised. When serum was dialyzed against an ordinary buffer solution, unbound cortisol% increased with the time of dialysis, accompanying a progressive decrease in serum protein concentration. These changes seemed to be due to the difference of colloidal osmotic pressure between the serum and the buffer. An addition of3% dextran in the buffer resulted in a stable equilibrium and both the unbound cortisol% and the protein concentration stayed unchanged after12hr of the dialysis. Therefore, the determination of unbound cortisol % was carried out by dialyzing serum against 3% dextran buffer for18hr and the method was called an isocolloidosmolar equilibrium dialysis. Dextran did not bind cortisol at all. Additions of graded amounts of cortisol in the dialysate caused significant increases in unbound cortisol % of a pooled serum. The ratio of unbound cortisol to albumin-bound cortisol was determined by the same dialysis system using heat-treated serum in the presence of nonradioactive cortisol. A combination of these two determinations enables to assess concentrations of unbound, albumin-bound and transcortin-bound cortisol in serum. Both intra-assay and inter-assay precisions of these methods were excellent (at most±4.65%).
In normal subjects (cortisol concentration: 7.1±2.5μg/100ml), 5.8±0.7% was present as unbound cortisol, 15.8±2.5% as albumin-bound and78.3±2.8% as transcortin-bound cortisol. Concentrations of all serum cortisol fractions increased with the course of pregnancy, however, relative increment of transcortin-bound cortisol was larger than that of total cortisol, while those of unbound and albumin-bound cortisol were far less. Percentages of transcortin-bound and albumin-bound cortisol changed in parallel to changes in concentrations of these cortisol-binding proteins during pregnancy.
pregnancy. The method requires a small amount of serum and is applicable to determine three cortisol fractions in individual sera.

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Injections of 2mg of progesterone into ovariectomized estrogen-primed rats significantly increased serum LH and FSH concentrations 3, 5 and 8hr later. Receptor blockers of noradrenaline (NA), dopamine (DA) or acetylcholine (ACH), phenoxybenzamine (20mg/kg body weight), pimozide (1mg/kg body weight) or atropine (700 mg/kg body weight), respectively, prevented the progesterone-induced gonadotropin release. On the other hand, none of them blocked the gonadotropin release following unilateral electrochemical stimulation (100μA for 60sec) of the medial preoptic area which occurred 0.5 and 1.5hr later, although pimozide or atropine reduced serum LH concentrations at 4.0hr after stimulation.
Furthermore, the sites of action of NA, DA and ACH with respect to LH release were examined by intracerebral implantation in ovariectomized estrogen-primed rats. DA or ACH, when implanted unilaterally into the medial preoptic area, induced a significant increase in serum LH 5hr later, whereas NA decreased LH levels. Implantations of NA or ACH into the bed nucleus of the stria terminalis or the medial amygdala increased serum LH although the effect of NA into the latter was not statistically significant. Only implantations of NA among the three substances into the lateral septum induced LH release.
These results suggest that all of NA, DA and ACH play stimulatory roles in the regulation of gonadotropin secretion, and that there are regional differences of their effectivenesses in releasing L H within the limbic-preoptic area.

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To determine whether the thyroid stimulating activity of IgG of patients with Graves' disease is associated with the reaction with a putative human thyroid antigen, the inactivation of the property of IgG to stimulate cAMP generation in human thyroid slices incubated in vitro was studied by pretreating the IgG with human thyroidal particulate fraction. In the preliminary experiment, it was demonstrated that to cause cAMP generation stimulation, on incubation period of120min is required to allow the IgG to penetrate the tissue. When human thyroid slices were incubated with normal IgG without or with pretreatment by human thyroid particulate fraction obtained from 100mg tissue, cAMP content in the slices was 142±25 or 138±26 f moles/mg, respectively, indicating that basal thyroidal cAMP levels were not influenced at all by normal IgG even after pretreatment with thyroid particulate fraction. When the slices were incubated with IgG of Graves' disease without or with the similar pretreatment, cAMP content was 320±31 or 140±25f moles/mg, respectively, demonstrating an almost complete inhibition of the activity of the IgG to cause cAMP generation stimulation.

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A 43 year old man with diabetes insipidus who showed panhypopituitarism and marked hypergammaglobulinemia due to histiocytosis X is reported. His low basal plasma adrenocorticotropin (ACTH) and growth hormone (GH) failed to respond to insulin-induced hypoglycemia. His basal serum thyroid hormone level was below normal and normal basal plasma thyrotropin (TSH) showed a delayed response with normal peak value to TSH-releasing hormone (TRH). Normal basal plasma pituitary gonadotropin also showed a delayed response with normal peak value to luteinizing hormone-releasing hormone (LH-RH). Suppression of plasma prolactin (PRL) by levodopa (1-dopa) was impaired and elevation of basal plasma PRL was noted at the second admission. These results, combined with diabetes insipidus, suggested that the panhypopituitarism in these patients was hypothalamic in origin.
The polyclonal hypergammaglobulinemia was characterized by elevated serum IgG and IgE levels which returned to normal after corticosteroid treatment with concomitant clinical improvement. Elevated serum IgE levels, tissue and peripheral eosinophilia, and the effectiveness of corticosteroid therapy support the hypothesis that some allergic mechanism may be involved in the pathogenesis of this disease.

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A refractoriness of livers from fetal and immature rats to glucocorticoid action on tyrosine aminotransferase has been found. To investigate the age-related responsiveness to the hormone, the binding capacity of liver cytosol receptor to cortisol and the nuclear binding to the cortisol-cytosol receptor complex were measured in relation to the change in ploidy pattern. The proportion of diploid nuclei in isolated liver cell nuclei decreased abruptly to give rise to polyploid, mainly tetraploid nuclei between 2 and 6 weeks after birth, while the binding capacity of liver cytosol to ³H-cortisol increased gradually. In livers from15week-old rats, the binding capacity decreased. Adrenalectomy 5 days prior to the experiment resulted in a clear increase in the cytosol binding capacity of livers from9week-old rats, while corticosterone supplement to the rats for 5 days caused a marked decrease. When isolated liver nuclei were incubated with ³H-cortisol receptor complex prepared from livers of 9 week-old rats, the acceptor capacity of whole nuclei preparation from mature rats such as 6-, 9-and 15-week-old rats was about two-fold as large as that from immature rats (newborn and2-week-old). Adrenalectomy resulted in a slight increase in the nuclear binding capacity of livers from 9 week-old rats, while corticosterone supplement suppressed more or less the capacity. In contrast to this, the acceptor capacity of whole liver nuclei from rats at various ages was nearly negligible when the cortisol-receptor complex was replaced by free ³H-cortisol.
When isolated nuclei from6week-old rats were fractionated into diploid and polyploid classes, the acceptor capacity of polyploid nuclei to the cortisol-receptor complex was much larger than that of diploid nuclei. A similar difference was also observed when the chromatin preparations from nuclei of both ploidy classes were used, but in fact no binding was observed when the stripped DNA was substituted for nuclei or chromatin preparations. Biochemical analyses of protein and DNA of liver nuclei from rats at various ages revealed that there was no difference between protein concentrations per DNA except that of newborn rats, but polyploid nuclei chromatin was lacking in some nonhistone protein and a part of H1histone. A possible implication of the ploidy change in the glucocorticoid action was discussed in relation to the present findings.

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