In order to study the secretory mechanism of human placental lactogen (hPL : hCS) from trophoblastic tissues, tissue culture and new placental perifusion systems were developed and used to clarify the effect of various substances on the secretion of hPL. These substances were (1), metal ions ([Ca²⁺], [K⁺], [Mg²⁺], [Na⁺]); (2) growth hormone and prolactin releasing or inhibiting factors (arginine, TRH, somatostatin, dopamine); (3) LHRH, dibutyryl cyclic-AMP which stimulates hCG secretion; (4) prostaglandins F_2α, and E₂, bradykinin; (5) EGF and insulin which have the receptors in the placenta; (6) glucose. It was found that most of the substances examined had no effect on the secretion of hPL, except dopamine and glucose. The effect of dopamine in the tissue culture system is dosedependent. At high concentrations dopamine slightly inhibited hPL secretion (5mM; 38.6 ± 15.0 and 10mM; 35.7 ± 19.0μg/g wet tissue) compared with the control (63.2 ± 29.8μg/g wet tissue).However, these effects may be due to the deviation of pH in the medium from the direct addition of dopamine hydrochloride. At a low concentration (1mM) it was observed to have a rather stimulatory effect (125.7 ± 18.0ggig wet tissue, p<0.05), but in the perifusion system, this effect could not be observed. The addition of glucose in the perifusion system gave a slightly higher hPL secretion than that of the control. Perhaps this resulted from increased cell activity rather than a stimulatory effect.
An incorporation experiment of [³H] leucine was also carried out to study the biosynthesis of hPL. Newly synthesized ([³H] -labelled) hPL was secreted into the medium within two hours. Furthermore, a labelled larger molecular weight substance together with the tritiated hPL was also observed on a Sephadex G-100 gel chromatography. These labelled substances were immunoprecipitatable using an anti-hPL serum, indicating that the substances contain the same immunological determinants. This result indicates that the larger molecular substance may represent the biosynthetic precursor or the aggregate of hPL.
These data indicate that the secretion of hPL has a unique mechanism, different from GH and PRL, and may be self regulated.

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Calcium binding protein (CaBP) has been reported to be involved in absorbing calcium in the duodenum. There is some evidence to show that active transport of calcium ions occurs from mother to fetus in the human placenta. The presence of CaBP in the human placenta was examined according to Taylor and Wasserman's method, extracting the vitamin D-dependent CaBP in the duodenum, and the character of CaBP was then studied. 38,000 × g supernatant of the villous homogenate of the human placenta was heated at 60°C for 5 mins to neutralize the calcium binding activity resulting from the blood. The fractions containing CaBP were separated and partially purified on Sephadex G-100 and DEAE cellulose columns. The CaBP fractions were further purified by preparative agar electrophoresis. The Chelex-100-resin method was used for the detection of calcium binding activity in the eluates and for calculation of Kd values and of calcium binding sites. The molecular weights were determined by electrophoresis of SDS-polyacrylamide gel. Three CaBPs were found in the human placenta. The molecular weight, Kd (μM) and the binding activity (Ca mol/CaBP mol) of the three placental CaBPs were CaBP-Peak I : 82,000, 0.64, 0.52; Peak II : 12,000, 0.67, 1.10; Peak III : 8,500, 0.75, 2.08, respectively. The tyrosine residue of CaBP has been shown to act as an important binding site in that removal of calcium ions from CaBP generates a blue-shifted phenomenon in the ultraviolet difference spectrum between 270 and 300nm. However no difference spectrum was observed with the placental CaBPs. This result was further confirmed by amino acid analysis which showed that none of the placental CaBPs contained tyrosine, tryptophan, half-cystine and proline, which are usually found in most proteins. The circular dichronism (CD) spectrum of CaBP-Peak I in the far ultraviolet range showed two negative bands at 222 and 207nm (α-helix structure), and removal of calcium ions caused no difference spectrum. CD spectra of CaBP-Peak II and III in the far ultraviolet range revealed random coil structures in the presence and absence of bound calcium ions. These findings indicate that the calcium binding mechanism of the placental CaBPs should be different from that of the others. In this study, three kinds of human placental CaBP newly isolated were characterized according to molecular weights, Kd values and binding activities and clarified as having an amino acid composition quite different from the CaBPs already reported.

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The responses of immunoreactive free α-subunit of glycoprotein hormones to LH-RH administration were studied in normal men and women, and in patients with hypergonadotropic hypogonadism, hypogonadotropic hypogonadism, trophoblastic disease and isolated ectopic α-subunit producing tumor.
In patients with hypergonadotropic hypergonadism, basal levels of serum α-subunit were elevated and the responses to LH-RH were also excessive compared to those of normal men and women. Conversely, in hypogonadotropic hypogonadism, basal levels of α-subunit were significantly low and its response to LH-RH was barely detectable.
The response of α-subunit to constant intravenous infusion of LH-RH (1μg/kg/h) was studied in 4 normal men. Both LH and α-subunit revealed biphasic patterns of elevation. Its releasing pattern suggests the possibility that two pools of gonadotropin are involved in the production and secretion of α-subunit.
In patients with trophoblastic disease secreting low levels of hCG (18mIU/ml), the responses of α-subunit as well as pituitary gonadotropin to LH-RH were normal. However, in cases of high concentrations of hCG (1000mIU/ml), the responses of α-subunit and gonadotropin were suppressed.
After the administration of LH-RH to a patient with an isolated ectopic α-subunit producing tumor, the serum concentration of pituitary gonadotropin increased within the normal range, although that of α-subunit did not show a significant change.
These results suggest that the production of α-subunit by tumors may be autonomous in contrast with a regulatory production in the pituitary.

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Urinary estriol-16-glucosiduronate (E₃-16-G) in early pregnancy was measured by a direct radioimmunoassay which was highly specific for the compound and did not require hydrolysis or chromatography. The level of E₃-16-G in normal pregnant women increased as pregnancy progressed and was over the level in non-pregnant women after 6 weeks gestation. The levels were lower in complicated pregnancies, namely threatened abortion, intra uterine fetal death, ectopic pregnancy, anencephalus and so on. Decreases of urinary E₃-16-G in threatened abortion were more rapid than those of urinary hCG, which was used as a useful prognostic indicator in those cases. These results suggested the possibility that the fetus played some role in the production of E₃-16-G in early pregnancy. In order to clarify the role of the fetus in the formation of E₃-16-G, it was measured in the maternal peripheral vein, uterine artery and vein, and in the fetal tissue at hysterectomy in complicated pregnancies (three cases). These results also suggested the possibility that E₃-16-G was produced by the fetus. In the in vitro experiments where ¹⁴C-E₃ was incubated with various tissues in early pregnancy, it was demonstrated that ¹⁴C-E₃ was conjugated to be E₃-16-G by homogenate of the fetal tissue in 8 weeks gestation, and by the liver and by the kidney in 13 weeks gestation but not by chorionic tissue. These results indicated that the conjugation process of E₃, though perhaps not entirely, proceeds in the fetus and the urinary determination of E₃-16-G might give us good information about the fetus in pregnant women.

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The interaction of LH to the receptor in a 2000 × g pellet of pseudopregnant rat ovary has been studied. The binding of ¹²⁵I-LH to the receptor was a highly specific and saturable process which was dependent upon time and tissue concentration. Maximum hormonereceptor binding was obtained within 30 min. in the 2000 × g pellet at 37°C. Displacement of binding of ¹²⁵I-LH by non-labeled hCG in vitro demonstrated that 10ng/ml of non-labeled hCG caused approximately 80% inhibition of binding of labeled hCG, and 10,000ng/ml of non-labeled hCG caused complete inhibition of the binding. Insulin and prolactin produced virtually no inhibition. Injection of 200IU hCG in vivo competitively inhibited the binding of ¹²⁵I-LH to the receptor. The effect of different enzymatic preparations on the binding of the hormone-receptor complex was investigated. Proteolytic enzyme and phospholipase D damaged the binding of the LH-receptor complex. An inhibitor of the binding for LH receptor was found in the supernatant of a 30,000 × g fraction of pseudopregnant rat ovary. The supernatant of the 30,000 × g fraction of pseudopregnant rat ovary apparently inhibited the in vitro binding of ¹²⁵I-LH to LH receptor. The dissociation constant (Kd) measured at 37°C obtained a value of 4.2 × 10^-10, and the number of binding sites was 55.6 fmol/mg protein in the 2000 × g pellet for LH receptor assay.
The mechanism of PGF₂α on the luteal function was investigated by measuring serum progesterone, serum PGF²α and the LH receptor of rat corpora lutea. Twenty-five day old female S-D rats treated with 50IU PMS followed by an injection of 25IU hCG 56 hours later was used. LH receptor was prepared with the 2000 × g pellet of the homogenate of the superovulated rat ovary. Extraction of PGF₂α was prepared by the Jaffe method, and PGF₂α was assayed by RIA. Administration of 200μg PGF₂α on the 7th day after hCG treatment resulted in a rapid and marked decrease in LH receptor and serum progesterone levels, while serum LH was below the detectable range. The affinity of LH receptor to LH (Kd=4.2 × 10^-10) was not changed, but the number of binding sites in the ovaries of PGF₂α treated rats was about 1/4 of the control group. When either indomethacin or alclofenac was administered to the rats intraperitoneally every day from the 10th to 16th day after hCG treatment, the levels of LH receptor and serum progesterone on the 16th day were still higher than in the controls. Administration of 200μg progesterone, 100IU hCG or 10μg E₂-17β subcutaneously from the 4th to the 7th day after hCG treatment increased serum PGF₂α level. The concentration of PGF₂α in the hysterectomized pseudopregnant rats which received 200μg progesterone was not increased in comparison to the non-hysterectomized pseudopregnant group on the 7th day after hCG treatment. These results indicated that PGF₂α, which was produced in the uterus by progesterone or estrogen administration, acted in to decrease the number of binding sites of LH receptor, resulting luteolysis.

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The influences of exogenous hCG on rat (pseudopregnant and pregnant) ovarian hCG receptors and the presence of hCG-like material in the rat placenta were investigated.
Ovarian hCG receptors in immature rats induced by PMS-hCG priming maintained a binding activity for about 20 days after hCG injection.
The administration of hCG to pseudopregnant rats caused time and dose related changes in ovarian ¹²⁵I-hCG binding percentage and the concentration of serum progesterone. The injection of hCG produced a slight decrease in the binding percentage and was followed by a marked decrease. The measurement of ovarian tissue hCG concentration by radioimmunoassay suggested that the initial loss of binding percentage was associated with the occupancy of receptors by the exogenous hCG, but that the major loss of binding percentage might not be attributed to the receptor occupancy. The active process might be involved in the mechanism of receptor loss. The progesterone concentration in sera had a tendency to increase after the injection of 251U hCG, while it decreased after the injection of 1251U hCG.
The ¹²⁵I-hCG binding percentage to ovaries and the serum concentration of progesterone reached their maximal values from mid to late pregnancy and declined as parturition approached.
After the injection of hCG into pregnant rats, the binding percentage rapidly decreased and the serum concentration of progesterone increased. Thereafter the binding percentage recovered on the 4th day after the injection of hCG. On that day, though the concentration of hCG in ovarian tissue was barely detectable, the binding percentage was significantly low compared to the control level.
The rat placenta (8th to 21st day of pregnancy) contained hCG-like activities, but they could not be detected in peripheral blood.
In the treatment of hCG-antibody in pregnant rats, the binding percentage did not change but the serum concentration of progesterone showed a tendency to decrease to the control level.
These studies show that hCG receptors exist as free sites in pregnant and pseudopregnant rat ovaries and may be regulated by down regulation after the treatment of large amounts of exogenous hCG. We found that hCG-like material in rat placenta determined by radioimmunoassay might serve as a luteotropin during pregnancy.

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Although the tuberoinfundibular dopamine (DA) system has been well documented to play a role in the regulation of prolactin (PRL) secretion, conflicting data have been presented regarding the other pituitary hormones, especially with regard to gonadotropin and growth hormone (GH) secretion. The site of DA action on gonadotropin secretion also remains obscure. The purpose of the present study was to investigate the effect and site of action of DA and, in addition, to examine the effect of a DA receptor antagonist, metoclopramide (MCP), on pituitary LH, FSH, PRL and GH secretion in women.
Dopamine HCl diluted in normal saline was infused at a rate of 4μg/kg/min for 180 min to 6 bilaterally ovariectomized women and 6 women with hyperprolactinemic amenorrhea (H-A). LH-RH (100μg) and TRH (500μg) were administered intravenously one month before and during the DA infusion (4μg/kg/min for 240 min, LH-RH and TRH being injected at 120 min) to 5 agonadal women. MCP (10mg) was injected intravenously to 5 agonadal women and 5 women with H-A. Blood samples were obtained through an indwelling venous catheter at 20 min intervals during the experiments. Serum LH, FSH, PRL and GH were measured by a double antibody RIA. Statistical analyses were performed by Student's paired or unpaired t-tests, as appropriate.
Serum LH levels significantly declined during the DA infusion and returned to basal level after the termination of infusion in both groups of subjects. The maximum decrement was about 9-fold greater in agonadal women than in women with H-A (19.5 ± 2.4 vs. 2.1 ± 0.5mIU/ml, p<0.001). FSH levels also significantly decreased during the DA infusion in each group, but the degree of decline in FSH was not so remarkable as in LH. A significant decrease and a significant rebound increase from baseline level in PRL were observed during the DA infusion and after the termination of infusion respectively in both groups, and the maximum decrement and maximum rebound increment were significantly greater in women with H-A than in agonadal women. That is to say that the basal LH, FSH and PRL levels were significantly correlated with the magnitude of decline in each hormone during the DA infusion, respectively (r= 0.837, p<0.001 for LH; r=0.794, p<0.01 for FSH; r= 0.917, p< 0.0001 for PRL). On the whole, DA failed to cause a consistent pattern of GH secretion in both groups, but it seemed likely that DA may exert a dual effect, such as stimulatory or inhibitory, on GH secretion according to its basal level. No significant changes occurred in LH and FSH responses to LH-RH during the DA infusion when compared with those to LH-RH alone in 5 agonadal women. In contrast, PRL response to TRH during the DA infusion was completely abolished and significantly different from that to TRH alone. MCP caused a rapid and remarkable rise (about 14-fold increase) in PRL levels in 5 agonadal women and, on the other hand, a transient and modest one (about 1.5-fold increase) in 5 women with H-A. A significant negative correlation between maximum increment following MCP injection and basal level in PRL was recognized in this experimental group (r=-0.886, p<0.001). In the other pituitary hormones, no observable changes, except for a transient but significant rise of LH in women with H-A, were produced by MCP administration.
These results suggest that DA exerts an inhibitory effect on pituitary LH, FSH and PRL secretion in proportion to each basal level. Furthermore, DA appears to inhibit gonadotropin secretion via suppressing LH-RH release, probably at the median eminence and to inhibit PRL secretion by a direct action on the anterior pituitary, because pituitary gonadotropin response to LH-RH was not altered even in the presence of DA, whereas PRL response to TRH was completely abolished by DA, and both the median eminence and anterior pituitary lie outside the blood-brain barrier,

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Plasma concentrations of vitamin D₃, 25-OH-D₃, 24, 25 (OH) ₂D₃ and 1, 25 (OH) ₂D₃ were measured in patients with various diseases using the multiple assay system previously reported. In patients with hyperparathyroidism, the plasma levels of 1, 25 (OH) ₂D₃ tended to increase, while the levels of 24, 25 (OH) ₂D₃ tended to decrease in many cases. On the other hand, plasma 1, 25 (OH) ₂D₃ levels were low, while 24, 25 (OH) ₂D₃ levels were high in patients with hypoparathyroidism. No significant differences in the levels of plasma D₃ derivatives among idiopathic-, postoperative-and pseudo-hypoparathyroidism were observed. In a majority of hemodialyzed patients with advanced renal failure who showed no overt bone changes on X-ray films, both the plasma 1, 25 (OH) ₂D₃ and 24, 25 (OH) ₂D₃ levels were distributed from normal to very low. In a majority of patients with osteoporosis and hypoparathyroidism, plasma 1, 25 (OH) ₂D₃ levels rose quickly after the administration of 1α-OH-D₃. In hypophosphatemic vitamin D resistent rickets, however, the response to the relatively large amounts of 1α-OH-D₂ was poor, although the concentrations of plasma 24, 25 (OH) ₂D₃ were elevated by the 1α-OH-D₃ administration in most of these cases.
The plasma 1, 25 (OH) ₂D₃ and 24, 25 (OH) ₂D₃ concentrations in patients with senile osteoporosis were distributed from lower to higher range than those of normal adults.
There was a relatively good correlation between plasma levels of 25-OH-D₃ and D₃ only in cases with plasma D₃ levels higher than 5ng/ml. In addition, a hyperbolic regression curve was obtained between the plasma D₃ and 25-OH-D₃ ratio. These results may indicate that a possible negative feedback homeostatic mechanism exists between plasma levels of D₃ and 25-OH-D₃, but only with low plasma levels of D₃.

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We report here on a patient who was unilaterally adrenalectomized for Cushing's syndrome, and who developed antibodies to 1-24 ACTH.
A 49-year-old nurse had undergone right adrenalectomy for adrenal adenoma. After surgery, she was treated with 0.5mg of 1-24 ACTH-Z together with glucocorticoid replacement therapy for 40 days. Thereafter she was given 0.25-0.5mg of ACTH-Z every other day for 4 months. ACTH-7_, was then stopped for a year but glucocorticoid therapy was continued. About one year prior to this admission, 1mg of ACTH-Z was again initiated 1 to 2 times a week. Glucocorticoid therapy was not withdrawn during the four years after adrenalectomy. She was admitted for the purpose of withdrawal of glucocorticoids. Her serum was found to bind labeled ACTH. This labeled ACTH was competitively displaced from binding by unlabeled hormones. Finally, reaction with specific antihuman Ig demonstrated an antibody of the IgG class. The titer of the antibodies gradually decreased after the discontinuation of ACTH-Z, but it is still present in measurable quantity in her serum. The clinical significance of the circulating anti-ACTH antibody in her serum is discussed.

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