We have investigated the effects of hormones and serum on glycosaminoglycan (GAG) synthesis, using cultured rat chondrocytes isolated from growing cartilage. Somatomedin A stimulated GAG synthesis at a physiological concentration, however in the case of insulin the dose required to stimulate GAG synthesis was 500 times as great as the physiological concentration. Parathyroid hormone also increased GAG synthesis. In contrast, hydrocortisone inhibited GAG synthesis at a pharmacological dosage. None of the following had any effect on GAG synthesis: epidermal growth factor, fibroblast growth factor, triiodothyronine, growth hormone, sex steroid or vitamin D₃.
Human serum up to a concentration of 1% stimulated GAG synthesis. Serum from patients with acromegaly stimulated GAG synthesis more than that from those with hypopituitarism.

View full abstract

The plasma calcium concentration, urinary calcium excretion, total bone calcium content and the rate of calcium uptake and release by tibias were measured in 6 groups of rats which were kept under various treatment protocols for 8 wk. Ovariectomy led to significant hypocalcemia and a decrease in bone calcium content the former being reversed towards normal by estrogen treatment. Chronic ethanol ingestion reduced the bone calcium content without apparent effect on plasma calcium in intact rat and further reduced (P<0.05) the bone calcium content in ovariectomized rats. The calcium uptake by the bone measured by ⁴⁵Ca injected ip at zero hour was the same among the 6 groups. When Ca was injected ip 17 hours earlier, the disappearance of plasma ⁴⁵Ca was found to be slower in ovariectomized and alcohol treated groups, indicating a higher rate of calcium movement from bone to plasma. Moreover, there was also an increase in the rate of calcium release by tibias after ovariectomy and chronic alcohol ingestion; the release was slightly greater in the condition of ovariectomy plus alcohol.
The present investigation thus, provides evidence that ethanol is likely to have a direct stimulatory effect on bone resorption as well as an indirect effect through disrupting ovarian function during chronic administration.

View full abstract

The renin-angiotensin system was studied in eight patients with Cushing's syndrome (four with adrenal adenoma and four with adrenal hyperplasia) and in five normal controls. Basal plasma renin activity (PRA) and aldosterone concentration (PAC) were similar in supine position among Cushing's syndrome due to adrenal adenoma (PRA ; 1.0±0.3 ng/ml/h, PAC ; 7.4±1.0 ng/dl, mean±SE), those due to adrenal hyperplasia (1.0±0.2, 6.9±0.8) and the controls (0.8±0.1, 6.4±0.4). The PRA after furosemide (1 mg/kg i.v.) and 120 min. upright posture stimulation was similar among Cushing's syndrome due to adrenal adenoma (2.2±0.7 ng/ml/h), those due to adrenal hyperplasia (2.6±1.7) and the controls (2.5±1.2). However, the PAC response after the stimulation in Cushing's syndrome due to adrenal hyperplasia (7.1±1.2ng/dl) was significantly lower than that in the controls (17.5±2.1)(p<0.01), although there was no significant difference between the PAC response in Cushing's syndrome due to adrenal adenoma (12.6±1.0) and the controls. These results indicate that PAC response to furosemide and upright pasture stimulation might be suppressed in Cushing's syndrome due to adrenal hyperplasia.

View full abstract

Cultured human urinary bladder carcinoma cells (JTC-32) were used to investigate the regulation of insulin receptors by dexamethasone. When the cells were preincubated with dexamethasone at 37°C, insulin binding sites increased up to 24 h. A large increase in insulin binding sites took place for 14 h of preincubation with dexamethasone. At lower concentrations of dexamethasone (<1 nM), no significant increase in insulin binding sites was observed, but the maximal increase was observed at more than 10 nM. Scatchard plots showed that dexamethasone increased the number of high affinity insulin binding sites (2.8 fold) without any change in the apparent equilibrium constant in JTC-32 cells. In addition, this steroid hormone also increased the number of low affinity insulin binding sites (1.6 fold) with a small change in the apparent equilibrium constant. Although insulin and dexamethasone did not affect the number of cells or the amount of cellular proteins per dish, dexamethasone plus insulin slightly increased them.

View full abstract

We have previously reported on the differences in physical and chemical characteristics between the high-molecular weight renin substrate (HMS>150, 000 daltons) and the normal substrate (NMS= 60, 000).
In this study, the kinetic constants were determined in both HMS and NMS which were prepared by gel exclusion chromatography from the plasma of pregnant women at term.
Renin substrate (angiotensinogen) levels were expressed by radioimmunoassay of angiotensin I after incubation of samples with added semi-purified human kidney renin in the presence of angiotensinase inhibitors.
The kinetic constants (Km and Vmax) were determined by the method of Lineweaver-Burk plots and also the method of Wilkinson. The Km for the HMS was 1.79μg angiotensin I equivalents (AIeq)/ml and the Vmax= 41.2 ngAleq/ml/h, and the Km for the NMS was 3.52μgAleq/ml and the Vmax = 138 ng/ml/h.
When adding small amounts of the HMS to the NMS, the production of angiotensin I was found to increase more than that in the NMS alone.
It was also observed that the renin substrate reactivities of the plasma of pregnant women, which contained small amounts of the HMS, were higher than that found in the plasma of normotensive women not taking oral contraceptives.
It is suggested that the existence of small amounts of the HMS may therefore contribute to the elevation in blood pressure under the influence of estrogens.

View full abstract

A specific, sensitive and simple radioimmunoassay (RIA) system for human transcortin was developed. A highly purified transcortin was prepared from pooled human serum by the following four successive steps ammonium sulfate fractionation, cortisol-Sepharose column chromatography, Ultragel AcAA column chromatography and hydroxylapatite column chromatography. Anti-transcortin antibody was raised by immunizing rabbits The RIA employing ¹²⁵I-labeled transcortin preparation and polyethylene glycol solution for separation of free and bound-form was sensitive to transcortin in concentrations as low as 10 ng/ml. This RIA was reliable in the tests of dilution, reproducibility and recovery. The presence of cortisol does not interfere with the assay. Also a test of cross reactivity revealed that the system was not influenced by human serum albumin in a concentration 10, 000 times that of transcortin.The transcortin concentrations determined by the RIA (Y) and those by the conventional steroid-binding assay (X) revealed a good correlation (Y=1.01 X+6.25) in normal serum, and the immunoreactivity and steroid binding activity revealed a good correlation in heat and acid-inactivated transcortin.
With some total cortisol concentrations given, the transcortin concentrations were inversely correlated with protein-unbound cortisol concentrations. The present assay is useful not only for biochemical research but also for clinical studies, in which the determination of transcortin makes it possible to evaluate the concentration of protein-unbound cortisol which is the physiologically active fraction in serum.

View full abstract

Immunoreactive β-endorphin (β-EP) in the ventricular fluid of six carcinomatous patients was measured using a specific radioimmunoassay. The subjects were undergoing a surgical procedure for relief of chronic intractable pain. This procedure involved the focal stimulation and coagulation of the posteromedial hypothalamus. Samples of ventricular fluid were collected before and after the stimulation and serially after the coagulation. Prior to stimulation, β-EP-like immunoreactivity (β-EP-LI) was below 200 pg/ml. In all of the six patients with pain relief, electrical stimulation led to a marked increase in immunoreactive β-EP. In three patients β-EP levels remained high after electrical coagulation for 6-24 hrs. These results suggest that β-EP-like material, released into the ventricular fluid, may contribute to the initial pain blockade that results from stimulation and coagulation of the posteromedial hypothalamus.

View full abstract

The method of pulsatile administration of gonadotropin-releasing hormone (Gn-RH) has been proven as a useful means for induction of ovulation in anovulatory women. In our series of clinical trials, 23 out of 29 anovulatory patients ovulated with pulsatile administration of Gn-RH. Seven patients who ovulated volunteered for the present study with daily hormonal analysis and follicular sonometory.
Two patients have oligomenorrhea, 3 patients secondary amenorrhea-1 st grade (the sole administration of gestagen required for withdrawal bleeding) and the remaining 2 patients secondary amenorrhea-2nd grade (the combined administration of estrogen and gestagen required for withdrawal bleeding). A diagnosis of hyperprolactinemia was made for one patient with secondary amenorrhea-1 st grade. Pulsatile administration of Gn-RH was performed by the use of a self-administered infuser. The infuser was connected to an i.v. indwelling catheter via a specially designed blood backflow eliminater. Five μg or less of Gn-RH was given every 2 hr from 07:00 to 23:00 hr daily. Five patients received HCG during the preovulatory period. In one patient, a short term treatment of HMG was added to Gn-RHtreatment.
Follicular sonometry revealed the development of a single dominant follicle which reached between 20 and 28 mm (23.7±0.12 mm, mean±S.E.) in diameter at the preovulatory period. Disappearance of a dominant follicle was recongnized in the early luteal phase. Characteristic increases in estradiol were recognized concomitantly wtih the development of a dominant follicle. Progesterone levels after ovulation were within the limits of its normal “luteal phase” rise.
The present data suggest that pulsatile administration of low dose Gn-RH with nocturnal interruption of treatment is effective for normal progress of follicular development in various types of anovulatory patients, culminating in single ovulation. This paper includes the discussion on our method which may be responsible for a high success rate of ovulation induction.

View full abstract

In a 26-year-old male with 17α-hydroxylase deficiency, endocrinological examinations were performed not only after, but also before the onset of clinical signs and symptoms. In addition, the pathophysiological role of the renal kallikrein-kinin system was investigated in this patient.
In spite of the fact that this disease is congenital, in the mechanism of its onset, this patient had a period of endocrinological normality before onset that is, 9 months before onset, both ACTH and cortisol were within the normal range, although the former would be significantly higher and the latter significantly lower than normal values after the onset. In this case, both urinary kallikrein and kininase excretions abnormally increased and then returned to normal after dexamethasone treatment.

View full abstract

The isolated cells were obtained from hog thyroid glands treated with dispase. More than 95 % of the cells obtained were intact and viable immediately after preparation, and the cell viability did not change during incubation in the experimental conditions.
ATP added to the external medium of whole cell suspensions was hydrolyzed in the presence of various divalent cations, especially Mg, and the rate of hydrolysis of ATP was not significantly different between the Mg-ion system and the completed ion system (Mg+ Na +K). When whole cell suspensions were disrupted with homogenizer, the hydrolysis of ATP was markedley increased by adding Na plus K. But there was no difference in the Mg-ion system between cell homogenates and whole cell suspensions.
ADP, AMP and adenosine as reaction products were found in the reaction mixture which resulted from the hydrolysis of ATP by whole cell suspensions.
Our data suggest that Mg-ATPase in the thyroidal isolated cells is an ectoenzyme whose active site (s) are exposed to the external surface of plasma membrane, and that ATP is finally hydrolyzed to adenosine via ADP and AMP by the enzyme (s).

View full abstract

It was demonstrated that thyrotropin-releasing hormone (TRH) elicited a paradoxical increase in basal GH levels in cancer patients.
Out of 94 cancer patients, 50 were found to be GH responders and this phenomenon was more frequently recognized in female than in male cancer patients. In cancer patients under 59 years of age, the GH response to TRH was significantly greater in females than in males, although there was no sex difference in the GH response in patients above 60 years of age. In female cancer patients, the GH response to TRH was significantly greater in patients under 59 years of age than in patients above 60 years of age, while there was no age difference in the GH response in male cancer patients.
It was concluded that paradoxical responses of serum GH to TRH were recognized in 53 per cent of cancer patients and were more frequently observed in female than in male cancer patients.

View full abstract

As compared with the pre-separated thymic lymphocyte (TL) population, a marked reduction of insulin binding was found in the two fractions [supernatant lymphocytes (SL) and precipitate lymphocytes (PL)] separated by low-speed differential centrifugation from the TL population. The reduction in insulin binding to SL or PL appeared to be due to a decrease in the affinity rather than a decrease in the number of insulin receptors. The reduced insulin binding was enhanced to near the level of the TL population when SL and PL were mixed and then incubated for 90 min at 37°C. Moreover, when the cell-free supernatant from the mixed and incubated fraction was added to SL and PL respectively, the insulin binding to each fraction was found to be enhanced.

View full abstract