Biological and immunological measurements of human luteinizing hormone (hLH) were performed with plasma samples obtained by continuous withdrawal over a 10-hour period in various phases of the normal menstrual cycle.
A pulsatile pattern of hLH discharge was demonstrated by both the in vitro bioassay and radioimmunoassay throughout the menstrual cycle, although the frequency and the amplitude of the pulse differed according to the menstrual stage. The decreased half disappearance time of immunoreactive hLH (83.7 min with 95% confidence limits at 64.3 and 103.1 min), in comparison with that of bioactive hLH (122.9 (98.2: 147.6) min), and the rapid recovery of the depressed biological to immunological activity ratio (B/I ratio) at the peak of the pulse (0.87 (0.77: 0.96)) in the periovulatory phase indicated the concomitant discharge of hLH subunits with hLH. The differences in the half disappearance times of bioactive hLH in the follicular and luteal phases (103.6 (94.7: 112.5) min and 87.2 (72.5: 101.9) min, respectively) and in the B/I ratios at the peaks of the pulses in these two phases (1.17 (1.05: 1.30) and 1.03 (0.94: 1.12), respectively) suggested the selective secretion of hLH subpopulations with various biological and immunological potencies and with various clearance rates. The significantly depressed B/I ratios at the trough in the luteal phase (0.58 (0.44: 0.76)) were sufficient to question the overall reliability of the hLH-RIA procedure employed, especially when applied to low hLH levels, even after several methodological improvements.

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Hemoglobin A_I (HbA_I) was studied in 21 cases of hyperthyroidism (H) and 33 control subjects (C).
The correlation equations between fasting blood glucose (FBG, mg/dl, x) and HbA_I (%, y) in the H and C groups were: y=0.07x+1.26 (r=0.5, p<0.05) and y=0.048x+2.23 (r=0.94, p<p.001), respectively. Five cases in the H group showed a higher level of HbAI far outside the confidence of 5% level of significance calculated from correlation equation for the C group. The correlation equation for the sum of blood glucose (ΣBG, mg/dl, x) during GTT and HbA_I (%, y) in the H group was y=0.003x+5.3 (r=0.64, p<0.01); in the C group y=0.005x+3.1 (r=0.88, p<0.001).ΣBG and HbA_I showed a close correlation in both groups. According to the results of the GTT, the subjects, either C or H group, were divided into three sub-groups: normal (n), borderline (b) and diabetic (d). The correlation equation between triiodothyronine (T₃, ng/dl, x) and HbA_I (%, y) in the H group was calculated as y=0.003x+6.49 (r=0.58, p<0.05). T₃ in the n subgroup was 331±90 ng/dl, in the b subgroup 362±166ng/dl and in the d subgroup 621±227ng/dl, and there was a statistical difference between the b and d groups.

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The release of somatomedin from perfused livers of normal and hypophysectomized (hypox) rats has been studied. Somatomedin was determined by the radioreceptor assay for somatomedin A. The release of somatomedin in the perfusates of normal livers was observed during the initial 30minutes and maintained during the second and third 30 minutes of recirculation. The addition of insulin at a concentration of 10mU/ml to the perfusates caused a significant increase in the release of somatomedin in the 1st, 2nd and 3rd 30-minute recirculatory perfusates. The addition of human growth hormone (25μg/ml) and/or insulin (1mU/ml) to the perfusates caused a significant increase in somatomedin after 120minutes of recirculation. Hypox rats released amounts of somatomedin in perfusates similar to those of normal rats. The effect of insulin on the release of somatomedin was greater in hypox rats than that observed in normal rats.
The molecular size of the somatomedin released in the perfusate was greater than that of purified somatomedin, suggesting that this somatomedin is bound to carrier proteins.

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The effects of intravenous salmon calcitonin on tissue carbohydrate and redox metabolism were studied in rat kidney in situ and were compared to the effects of EGTA infusion. Calcitonin (0.8 MRC U, in bous) caused, after 10-20 min, a reductive shift of the cytosolic NAD redox pair, and an oxidative shift of the mitochondrial NAD redox pair. It also caused a stimulation of glycolysis with the cross-over between phosphoenolpyruvate and pyruvate. These results were reproduced by treatments of hypocalcemia with EGTA or with thyroparathyroidectomy. Most of the effects were opposite in direction to the reported effects of CaCl₂ and parathyroid hormone. The above results suggest that in kidney calcitonin causes a stimulation of pyruvate kinase, and/or inhibition of phosphoenolpyruvate carboxykinase, and that the effects are related to changes in the intracellular calcium.

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Antiserum to prolactin (PRL) of bullfrog (f), Rana catesbeiana, was produced by immunizing rabbits with the highly purified fPRL obtained from adenohypophyses of adult bullfrogs. In Ouchterlony's agar double diffusion test, a single precipitin line was produced between the fPRL antiserum and fPRL or the bullfrog pituitary extract. No precipitin line was produced between the antiserum and ovine PRL, bovine PRL, ovine growth hormone (GH) or fGH. The acetone-dried powder of bullfrog pituitary glands which had been incubated with the fPRL antiserum had much less potency in promoting collagen synthesis of the tail fin of bullfrog tadpoles than that incubated with normal rabbit serum. Histological studies on bullfrog adenohypophyses revealed that the cells that immunologically reacted with the antiserum to fPRL were erythrosinophilic.
The antiserum to fPRL was used to develop a radioimmunoassay (RIA) in which fPRL and ¹²⁵I-fPRL were employed as the standard and the radioligand, respectively. Several dilutions of plasma of both adult and larval bullfrogs yielded dose-response curves which were parallel to the standard curve. Bovine PRL, ovine PRL, ovine GH and fGH did not react in this assay. Plasma from hypophysectomized bullfrogs had no detectable immunoreactive prolactin. Pituitary homogenates of Rana catesbeiana and Bufo bufo japonicus gave inhibition curves which were parallel to the standard. Pituitary homogenates of Xenopus laevis and Hynobius tokyoensis gave inhibition curves which did not parallel the standard. RIA of plasma PRL in larval bullfrogs of various developmental stages were performed. Average concentrations of prolactin during premetamorphosis (st. X), prometamorphosis (st. XVI-XIX) and at early climax stage (st. XX-XXII) were 18, 21-25 and 27-35ng/ml, respectively. At advanced climax stage PRL levels were quite high. Average values at stages XXIII, XXIV and XXV were 98, 169 and 116ng/ml, respectively. The significance of PRL levels in relation to metamorphosis is discussed.

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An assay for detection of serum IgG binding to thyroid membrane using ¹²⁵I-Protein A was examined. The test serum is incubated with purified thyroid membranes, and the IgG bound to the membrane is detected by its interaction with ¹²⁵I-Protein A.
Most of the bound IgG is not bound to the TSH receptor, because TSH does not induce any appreciable decrease in the binding of the IgG to thyroid membranes.
Increased serum IgG binding to thyroid membrane is found in most patients with Graves' disease and Hashimoto's thyroiditis, but not in patients with thyroid cancer or simple goiter. Many sera with positive binding activity showed positive microsomal antibody. Serum IgG binding to thyroid membrane in Graves' disease correlates neither with LATS activity nor thyroglobulin antibody. This finding suggests that TSH receptor is not involved in the reaction.
The assay method is useful for measuring the binding immunoglobulin for thyroid membrane that is frequently increased in autoimmune thyroid disease, and the present data provide further support to the concept that thyroid autoimmune disorders are associated with antibodies to thyroid cell surface components.

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Nine healthy female controls and 10 hyperthyroid female patients were studied. The intravenous administration of 200 mg cimetidine, an H₂-receptor antagonist, was followed by a significant and marked rise in serum prolactin levels in all control subjects. There was no significant difference in serum PRL response to cimetidine injection between the euthyroid controls and hyperthyroid patients. But max ΔPRL, the change from basal to peak values, is significantly lower in the hyperthyroid patients than in the controls. There was a significant negative correlation between max ΔPRL and serum T₄ or T₃ levels in hyperthyroid patients before and after treatment with MMI or PTU. It appears from our data that cimetidine induced PRL release was blunted in hyperthyroid patients.

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An unusual case of isolated ACTH deficiency with coexisting chronic thyroiditis in a 53-year-old man is reported. The patient was admitted with a 2-year history of generalized fatigue, a 13-kg weight loss, muscular weakness, and frequent hypotensive and hypoglycemic attacks.
On admission serum thyroxine and triiodothyronine were significantly elevated. Basal TSH concentration was not detected and TSH showed no response to TRH, but one month after replacement therapy with hydrocortisone it was shown that serum T₃, T₄ and TSH response were all within normal limits. Thyroid antibodies were positive and biopsy of the thyroid gland showed chronic thyroiditis.
Arginine and 1-Dopa provoked a subnormal rise in GH with a maximum of 5.6 ng/ml and 5.0, respectively. One month after treatment with hydrocortisone, GH response to 1-Dopa and arginine increased to the normal range. Prolactin response to TRH was normal and FSH response to LHRH was also normal. LH showed an exaggerated response to LHRH, although a normal response was revealed after treatment with hydrocortisone. We also presented a summary of 44 Japanese cases, 23 males (mean age; 46 yrs old) and 21 females (mean age; 48 yrs old), with isolated ACTH deficiency.

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The biochemical effect of a nicotinamide analogue, 6-aminonicotinamide (6-AN), on developing chick embryonic femur was studied. Growth of femur from 9-day-old embryos that had been exposed to 6-AN for 5 days in ovo was not stimulated by PTH in an in vitro culture system. PTH caused a much smaller increase in the cyclic-AMP content in 6-AN-treated femur than in control femur. However, dibutyryl cyclic-AMP stimulated growth of 6-AN-treated femur. A defect in response of 6-AN-treated femur to the growth stimulating action of PTH may explain the production of micromelia by 6-AN.

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A potent LH-RH agonist, des-Gly¹⁰-[D-Leu⁶]-LH-RH-ethylamide (TAP-144), administered to pregnant rats by the intravaginal and subcutaneous routes terminated pregnancy most effectively during days 7-11 of pregnancy. A daily administration of TAP-144 during days 7-10 was more effective by the intravaginal than by the subcutaneous route. When TAP-144 was administered twice a day during this period for one to three days, the effective dose became almost the same in the two routes. Complete termination of pregnancy occurred following a dose of 1μg/ 100 g body weight/a time by a single-day administration and 0.01μg/100 g body weight/a time by three days administrations. Previously we found that the serum concentration of TAP-144 after vaginal administration was lower but much longer-sustained than that after subcutaneous administration. Thus, in pregnancy termination, the prolonged duration of an effective concentration of TAP-144 seems to be more important than a transient high concentration. When rats were given effective doses of TAP-144 twice a day on day 9 by either route, ovulation occurred the next day (day 10) and the fetuses were aborted on day 11. Serum concentrations of progesterone and estradiol-17β dropped sharply. Estrus occurred on days 11-13, cycles were established thereafter, and ovulation occurred at regular intervals.

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In order to better understand the role of glucocorticoid receptor in the hormonal action of glucocorticoid, depletion and replenishment of the cytoplasmic receptor and kinetics of induction of tyrosine aminotransferase (TAT) in rat liver were examined after administration of dexamethasone (Dex) and prednisolone. The extent of the receptor depletion and the duration of the depletion period were dose-dependent and were well correlated not only with the biological potency of the steroids administered but also with the kinetics of TAT induction. A linear relationship between the amount of depleted receptor and the kinetics of TAT induction was observed. Pretreatment of animals with a large dose of Dex reduced the response of TAT induction by Dex which was administered 24 h later. This seems to be attributable, at least in part, to the incomplete replenishment of the receptor and also to the increased rate of replenishment of the receptor. The reduced affinity of the partially replenished receptor to glucocorticoid might also participate in lowering the response to the second injection. In conclusion, depletion and replenishment of the cytoplasmic glucocorticoid receptor appeared to be directly responsible for the physiological action of glucocorticoid.

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Fat cell TSH receptor-related antibodies were detected by immunoprecipitation of ¹²⁵I-TSH-receptor complexes and the nature of the antibodies was analyzed. To ¹²⁵I-TSH prebound to Triton-solubilized receptors from guinea pig fat tissues, 50μg of immunoglobulin G (IgG) was added and precipitation was effected by the addition of antihuman IgG.
Immunoprecipitation values in 13 patients with Graves' disease were significantly (p<0.05) higher than those in 11 normal subjects. No significant increase in the values was seen in 8 patients with Hashimoto's disease. No correlation was observed between immunoprecipitation values and titers of antimicrosomal and antithyroglobulin antibodies. Neither was there any correlation between the values and TSH-binding inhibitor immunoglobulins (TBII) detected by the radioreceptor assay. The IgG fractions positive for the immunoprecipitation antibody were found to be poor human thyroid stimulators (HTS) relative to their TBII activities. And a highly significant correlation was observed between TBII and HTS activities among IgGs without detectable antibody by immunoprecipitation (r=0.907; p<0.005; n=7). These findings 1) demonstrate that immunoprecipitation assay using fat cell TSH receptor may detect TSH receptor-related antibodies different from TBII in patients with Graves' disease and 2) suggest the antibodies may recognize determinants on the receptor or its vicinity that do not participate in the binding of TSH or thyroid stimulating antibody, and may interfere with thyroidal response to these stimulators.

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A radioreceptor assay of TSH using Triton-solubilized human thyroid receptors was applied to the detection of TSH-binding inhibitor immunoglobulins (TBII). In 26 untreated patients with Graves'disease, 20 (76.9%) were found positive in this assay, while 18 (69.2%) cases were found positive by the conventional assay using human thyroid particulate receptors. In 25 patients with goitrous Hashimoto's thyroiditis, 2 (8.0%) were found positive by the assay using soluble receptor, while 3 (12.0%) were found positive by the conventional one. A significant correlation was found between TBII activities in the patients with untreated Graves' disease detected by these two assay systems (r=0.69; n=26; p<0.001). These data support the concept that TBII are antibodies against the TSH receptor or its closely related structures.

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We established a sensitive RRA system for human FSH, employing PMS-treated immature rat ovary. The dissociation constant of the binding of the receptor preparation to NIAMDD human FSH-2 was 1.15×10^-10M. The standard curve was obtained with 0.2-25.6ng/tube of NIAMDD hFSH-2. Purified hLH, hTSH, and HCG had no significant effect on the binding. When the anterior pituitary homogenates obtained from humans were assayed by this system, the intra-assay and inter-assay coefficients of variation were 11.9% and 13.4% respectively, and the assay values correlated well with those obtained by RIA. This assay is applicable for the measurement of FSH in serum, when the nonspecific inhibitor effects of serum are compensated for by the addition of merthiolate and when FSH-free serum is used instead of the buffer for the standard curve. The intraassay, and inter-assay coefficients of variation were 9.31% and 19.7% respectively. The assay values correlated with those obtained by RIA under the same physiological state. The ratio of the assay values RRA/RIA, was dependent upon the physiological state, e.g. 6.29 in men, 3.84 and 4.18 in women at follicular and luteal phase respectively and 2.40 in menopausal women. During the menstrual cycle, our results showed that the value of RRA/RIA derived from serum did not change significantly.

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The effect of LH-RF on pituitary content of cyclic cytidine 3' 5' monophosphate (c-CMP), one of the newly detected cyclic nucleotides, was examined by the RIA procedure. In results showed that the pituitary content of c-CMP was lower than in various other organs of the rat. When incubated in vitro in the presence of 0.5 ng LH-RF/ml incubation medium, c-CMP content of the anterior pituitaries was reduced slightly, but the difference was not statistically significant when compared with the controls. No difference in c-CMP content was observed between the controls and the 5 ng LH-RF/ml group. C-CMP content in the rat anterior pituitary tissue did not change significantly during an in vitro time-course study (5, 15, and 30 min.) in the presence of 5 ng LH-RF/ml. In contrast, c-AMP content of the pituitary was significantly (P<0.05) elevated by the stimulation of 5 ng of LH-RF/ml at a 15 minutes of incubation. These data suggest that c-CMP content, unlike c-AMP, might not be changed significantly by hypothalamic gonadotropin releasing hormone.

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The present study evaluated the effect of sodium loading and sodium depletion on cAMP and cGMP content of rat aorta. Enhanced generation of prostacyclin (PGI₂) in aorta was noticed in sodium loading and sodium-depletion. As PGI₂ is potent vasoactive agent, the interaction between PGI₂ generation in the aorta and cyclic nucleotide accumulation in the aorta was investigated. Sodium depletion of rats induced the elevation of both cAMP and cGMP in the aorta and of cAMP in plasma. No change in cyclic nucleotides was noticed following sodium loading. These changes in cyclic nucleotides were felt to reflect the fluctuation of vasoactive agents under various sodium metabolism conditions. These observations may indicate that PGI₂ is not a major factor in the regulation of cyclic nucleotide metabolism under sodium loading and sodium depletion. The increased accumulation of cAMP and cGMP in rat aorta in sodium depletion may be due to the cumulative effects of PGI₂, catecholamine and probably angiotension II which are induced by sodium depletion.

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ACTH-induced steroidogenesis in isolated rat adrenal cells was markedly increased by commercially available preparations of calcitonin and hCG, but neither synthetic eel calcitonin nor highly purified hCG preparation had the same effect. The ACTH-potentiating activity was present in a variety of normal rat tissues including the cerebrum, the thyroid, the lung and the liver.

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Twice daily subcutaneous injections of 1 mg human placental lactogen for 1 week to female mice significantly suppressed the pituitary prolactin synthesis, which was measured by the in vitro incorporation of [¹⁴C] leucine into prolactin. On the other hand, 2 isologous pituitary grafting affected neither synthesis nor release of prolactin in the in situ pituitary even after 8 weeks, while the grafting resulted in the constant elevation of circulating prolactin to the proestrous level, the highest under normal physiological conditions, and induced marked mammary growth. Based on these observations, the significance of short-loop feedback of prolactin is discussed.

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The in vitro effects of the protein purified from the culture medium of Bordetella pertussis (pertussis toxin) on cyclic AMP levels in canine thyroid slices were studied. The toxin caused a three-fold increase in tissue cyclic AMP levels after 18h or longer incubation. Furthermore, when the slices were incubated with the toxin for 6h and then incubated without the toxin for further 12h, similar effects were observed. These results indicate that pertussis toxin elevates tissue cyclic AMP levels after a time lag in a manner different from other thyroid stimulators.

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The effect of a potent inhibitor of dopamine-β-hydroxylase, diethyldithiocarbamate (DDC) or fusaric acid, on the antidromically evoked potentials by electrical stimulation of the preoptic suprachiasmatic area (POSC) was examined. Thirty seven neurons in the ventrolateral part of medulla oblongata (VLMO) were successfully tested after DDC injection. Nineteen of them decreased in amplitude to approximately 50% of control levels at 120min after DDC injection, 20% at 180min. These 19 neurons were characterized by having a notch in the rising phase and taking a longer time to complete the initial deflection. After fusaric acid injection, ten neurons in the VLMO were successfully tested and four of them decreased in amplitude similar to DDC injection.
Effects of receptor blockers of noradrenaline, dopamine and serotonin on the single unit responses of the POSC neurons to the VLMO stimulation in proestrous rats were examined in order to know which of noradrenaline, dopamine or serotonin are involved in the neural transmission from the VLMO to the POSC. After the injection of phenoxybenzamine, an α-adrenergic receptor blocker, the rates of facilitatory and inhibitory responses in the POSC neurons to the VLMO stimulation were significantly decreased. Percentages of facilitatory and inhibitory responses were not changed significantly after injection of pimozide, a dopamineric receptor blocker, or methysergide, a serotonergic receptor blocker. These findings suggest that noradrenaline might be involved in the transmission of the VLMO stimulation effect on the POSC neurons and that VLMO noradrenaline-containing neurons send their axons directly to the POSC.

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