A bioassay method for the determination of ACTH in rat the serum was established. For the separation of ACTH from the serum, ACTH was adsorbed to Quso G 32 and then eluted with 40% acetone-1% acetic acid solution, and the eluate was evaporated at 45°C. The dried sample was incubated with isolated rat adrenal cell suspension and produced corticosterone was determined. The amount of ACTH was estimated from the standard dose-response curve. The minimum detectable amount of ACTH_1-24 added in the ACTH-free serum was 5 pg. Recovery and reproducibility of the assay were satisfactory for practical use.
The bioassay was applied to the determination of the ACTH level in the rat serum after morphine administration. After a 20mg/kg of morphine hydrochloride ip, ACTH concentration in the serum increased rapidly and its maximum level was observed 5min after the injection. The ACTH then decreased gradually, but it was still at a high level 60min after the injection. With an sc injection of the same amount of morphine, ACTH in the serum increased gradually and the maximum concentration was seen 60min after the injection. Despite the difference of time courses of ACTH concentration in the serum with different routes of morphine administration, the time courses and the maximum levels of corticosterone concentration were similar. An ACTH level in the pituitary was maintained at a pre-injection level even 60min after a 20mg/kg of morphine ip. In the experiment of morphinization, increasing doses of morphine were injected sc twice daily for 10 days. The ACTH concentration in the serum of morphinized rats did not increase with a test dose of 20mg/kg of morphine ip, but increased markedly with a 5 mg/kg of nalorphine hydrochloride ip. The ACTH level in the pituitary increased by about 40% in the morphinization.

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Tolbutamide was administered into the superior pancreaticoduodenal artery of anesthetized dogs. A prompt hyperinsulinemia was induced but the plasma gastrin increased gradually in the right gastroepiploic vein; acutely secreted endogenous insulin did not result in gastrin secretion. These findings suggest that gastrin is not released by insulin per se, but by decreased blood glucose.
By infusion of somatostatin into the superior pancreaticoduodenal artery, plasma gastrin in the antral vein and plasma insulin in the pancreatic vein decreased promptly, and the tolbutamide-and arginine-induced gastrin and insulin secretions were impaired. These inhibitory effects of somatostatin on the plasma gastrin and insulin secretion were quite clearly demonstrated in the right gastroepiploic vein and superior pancreaticoduodenal vein. The inhibitory effect of somatostatin was more pronounced on gastrin release than on insulin release.
It appears likely that the inhibitions of gastrin and insulin secretion by somatostatin were caused by direct action on the gastrin secretory cell and on the pancreatic beta cell. Somatostatin may play an important role in the entero-insular axis.

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The effects of synthetic neurotensin on plasma insulin and gastrin secretion were investigated in twelve normal and eight hypophysectomized (10th to 14th day post hypophysectomy) dogs. Neurotensin was injected rapidly or infused for 30 min into the superior pancreaticoduodenal artery, and plasma insulin and gastrin were measured by radioimmunoassay.
In normal dogs, rapid administration of neurotensin (10μg/kg body wt) brought about a mild hyperglycemia and a rapid and sharp increase of plasma insulin and gastrin levels. In the pancreatic vein, a biphasic insulin response was noted. The same secretory pattern of insulin was demonstrated during neurotensin (500ng/kg/min) infusion. In the antral vein, the plasma gastrin level increased rapidly to 425% from basal level within 2 min and returned nearly to the initial level within 10 min after a rapid administration of neurotensin.
In hypophysectomized dogs, the basal levels of plasma glucose, pancreatic insulin and antral gastrin were decreased to 53%, 9%, and 65% of normal dogs, respectively. Neurotensin has a little effect on insulin secretion, and a biphasic insulin secretory pattern observed in normal dogs was not demonstrated in hypophysectomized dogs. It seems likely that hypophysis may in part play a role in the neurotensin-induced insulin secretion. However, neurotensin-stimulated plasma gastrin concentrations were considerably maintained. Also, xenopsin (10μg/kg body wt) induced a mild hyperglycemia, hyperinsulinemia and hypergastrinemia, but its secretory activities were smaller than those of neurotensin.
Neurotensin appears to stimulate the insulin and gastrin secretion by the mode action of neurogenic vasodilatation or by the direct action of pancreatic beta cell and gastrin secretory cell of the gastric antrum. Neurotensin may play an important role in the entero-insular axis.

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The biosynthetic study utilizing ¹⁴C-eicosatrienoic acid or ¹⁴C-arachidonic acid as substrate was carried out with human fetal membranes and villi at term pregnancy. Prostaglandin synthetase activity was demonstrated in the homogenates of decidua, chorionic membrane, amniotic membrane and villi. The enzyme was localized in the microsomal fractions of decidua and amniotic membrane, of which relative activities were 5.1% and 3.1% to that of bovine seminal vesicle microsome, respectively. To achieve the maximal enzyme activity, GSH, Hb and 1-epinephrine must be present. The only type of prostaglandins found in our assay was only Ecompounds, which seemed to be due mainly to the cofactors employed. Production of PGE₂ in the microsomal fraction of either decidua or bovine seminal vesicle was inhibited by the 80, 000 × g supernatant fraction of decidua, which was shown to contain an inhibitory substance of heat-unstable and non-dialyzable nature. The significance of the experimental results in assessing the control of PG production in human decidua is discussed.

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Gel filtration of extracts of human chorionic tissues cultivated in vitro and of culture media has been performed. Heterogeneous immunoreactive forms of hCG and its subunits in tissue extracts were apparent. A void-volume immunoreactive peak of hCG was identified in extracts of chorionic tissues cultivated for a very short period. The immunoreactive void-volume material, which did not readily convert to the authentic form of hCG either spontaneously in refiltration or in 5 M guanidine, could not be distinguished immunologically from purified hCG and yielded an hCG-like component in trypsinization.
On gel filtration of chorionic tissue extracts, radioreceptor activity for hCG utilizing membrance fraction of pseudopregnant rat ovary always demonstrated a single peak in the region of ¹²⁵I-hCG used as column marker, indicating high receptor activity in the authentic form of hCG and low in the large immunologic species of hCG. Heterogeneity in the molecular size of bicactive hCG was not found with radioreceptor assay.
On the other hand, gel filtration profiles of chorionic tissue extracts and of culture media showed that the large immunologic species of hCG is a predominant form of the hormone in the chorionic tissue cultivated for a short period and the authentic form of hCG is a predominant form in the media. These data suggest that immunoreactive “big hCG” synthesized initially in placenta may be converted to the authentic form of hCG by trypsin-like enzyme and that the authentic form of hCG may be readily secreted into the media. These results support the proposal that hCG is also synthesized as prohormone in placenta. However, the biosynthetic process of hCG subunits remains yet to be resolved.

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Serum somatomedin A was determined by a radioreceptor assay in six patients with GH deficiency before and after im administration of GH in doses between 0.5 and 8mg. The mean basal value of somatomedin A, 0.36±0.02 U/ml (mean±SEM), was significantly lower than that of age-matched controls, 0.82±0.02 U/ml. There was no diurnal variation. Somatomedin A levels increased significantly eight hours after GH administration, remained elevated for 48 hours and returned to basal levels after 72 hours. The mean levels of somatomedin A found 24 hours after 1, 2, 4 and 8 mg of GH were 0.45±0.05, 0.68±0.10, 0.71±0.08 and 1.03±0.02 U/ml, respectively. A linear dose-response curve between the logarithmic dose of GH and the somatomedin A increase was found in three of six patients.

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Kinetic analyses of glucose-induced insulin release from the perfused rat pancreas and the perifused rat islets were performed by Lineweaver-Burk's and Hill's equations. All dose-response curves tested here were sigmoid with Km values of 4-5 mM and 8-9mM glucose, respectively in a K⁺-depleted and the normal media in the perfusion system, and with a Km value of 8-9mM glucose under either a square-wave or a slow-rise stimulation in the perifusion system. Each Lineweaver-Burk plot was represented as a parabolic profile and therefore each insulin secretory response seem to be n reaction orders (n>1) for perfusion and the perifusion with the normal medium, except for the perfusion with the K⁺-depleted medium. All Hill plots for the rate of insulin release from the perfused pancreas and perifused islets under all experimental conditions were demonstrated as straight lines with various slopes.
Each kinetic constant, e.g., Km (Michaelis constant), K (equilibrium constant) and n (Hill's constant or reaction order) was calculated from Hill's equation applied to the insulin secretory response, and particularly each Km value was very similar to that described above. It was concluded that Hill's method seemed to beb available for the kinetic analysis of the rate of glucose-induced insulin release.

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Estradiol-17β, estrone, estriol or diethylstilbesterol showed the increase in arginase activity of the dorsolateral prostate of intact adult male rats. Neither castration nor adrenalectomy modified the response of arginase activity by administration of estrogen. However, the arginase activity of the dorsolateral prostate of hypophysectomized male rats was significantly reduced regardless of estrogen treatment. On the other hand, almost the similar decrease was observed in the arginase activity of the ventral prostate from castrated, adrenalectomized or hypophysectomized rats, as well as from intact ones.
From these results, it was concluded that the increase in arginase activity of the dorsolateral prostate after estrogen treatment might be due to some indirect effects of estrogen, and that pituitary the gland seemed to be involved in this estrogenic effect.

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Serum LDH activity was found to be elevated in 16 out of 20 patients with Cushing's syndrome. This elevation was mainly due to increased LDH¹ and LDH², and correlated with the levels of urinary 17OHCS. When the glucocorticoid excess was corrected by surgery, elevated serum LDH levels tended to be normal. Thus, it was concluded that elevated serum LDH is a common finding in Cushing's syndrome and glucocorticoid excess may cause tl-e elevation of LDH by some undetermined mechanism.

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Female mice of the C3H/HeMs strain were given a single subcutaneous injection of various doses of testosterone propionate (TP) on Day 15 or 17 of fetal life or on the day of birth. Irreversible ovary-independent vaginal cornification and uterine stratification with or without squamous metaplasia were most frequently observed in mice given TP injections on Day 17 of fetal age. Vaginae of many mice given prenatal injection of higher doses of TP failed to form the orifice, whereas the orifice was always normally formed in neonatally injected mice. In contrast, it seemed probable that hypothalamic function was affected in less extent by prenatal treatment than by postnatal treatment. Of interest was that some mice with the vaginal lesion had normal reproductive capacity when they did not show uterine stratification.

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Occurrence of permanent proliferation and cornification of the vaginal epithelium in ovariectomized adult mice which had received neonatal injections of 20μg estradiol-17β(E²) was prevented by injections of 200IU vitamin A acetate (VA) given simultaneously with E². When neonatally E²-treated mice were parabiotically joined with ovariectomized mice treated neonatally with E² plus VA, the permanent vaginal changes were suppressed in the E² parabionts after the period of 97-110 days of union. These permanent changes were also suppressed by transplantation of vaginae from the E²-treated donors into the E² plus VA-treated hosts. A possibleme chanism for the degeneration of permanently changed vaginal epithelium is discussed.

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Male mice were subcutaneously injected with several gonadal hormones daily for 5 days in order to study their effects on hyaluronic acid (HA) content in the skin.
Increases in both total hexosamine and HA contents were in the following order: estradiol-17β(10μg)> estradiol-17α(10μg)> estradiol-17α(10μg)> testosterone (100μg)>control (p<0.001). NO Significant differences were noted between 5α-dinydrotestosterone (100μg) and control. Testosterone (100μg) or 5α-dihydrotestosterone (100μg) injected with estradiol-17β(10μg) blocked the increase in HA content produced by estradiol-17β alone.
Thus, it can be suggested that an increase in HA is not specific for estradiol-17β and that the action of testosterone or 5α-dihydrotestosterone is antagonistic to that of estradiol-17β in the male mouse skin.

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Subcutaneous injection of croton oil for stress or intraperitoneal injection of metyrapone was given to rats daily for 14 days after unilateral ovariectomy. Six-day adrenalectomized rats were unilaterally ovariectomized. The animals were sacrificed 15 days after unilateral ovariectomy. The weight of the ovary was expressed as that for 100g body weight.
At this time, the remaining ovary of the unilaterally ovariectomized control increased significantly in weight. In contrast, no weight increase was found in the remaining ovary of the stressed unilaterally ovariectomized animal, which could, however, respond well with a weight increase to the small amount of HCG injected daily for 4days before autopsy. Consequently, these results indicate that the stress may decrease gonadotropin secretion, leading to suppressing compensatory ovarian hypertrophy.
Metyrapone or adrenalectomy also abolished compensatory ovarian hypertrophy. Further, the remaining ovary of the same animals was shown to be able to take up preferentially ¹²⁵I-labelled HCG on the day of autopsy. Therefore, metyrapone or adrenalectomy may also reduce gonadotropin secretion to inhibit compensatory ovarian hypertrophy.
The mechanism involved in the stress, metyrapone or adrenalectomy-induced reduction of gonadotropin secretion was discussed.

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The effect of hypophysectomy on somatostatin (SRIF) content in the rat pancreas was investigated. SRIF content was determined by the specific radioimmunoassay developed by us.
Four weeks after hypophysectomy, the total SRIF content in the rat pancreas had not changed or decreased, but the content per mg wet weight as well as per mg protein had increased markedly and the content of islets obtained by collagenase digestion had showed a tendency to increase. The mechanism and the pathophysiological significance responsible for these changes remain to be determined, so further examination of this problem will be necessary.

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