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Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.

Surgically isolated pig thyroid glands, without parathyroid tissue, were perfused in situ with blood, the calcium concentration of which could be varied. Complete, measured collections of thyroid venous blood were achieved, and the thyrocalcitonin (TC) content of each sample was determined by intravenous bioassay in young rats. The time course of changes in perfusing calcium concentration was closely paralleled by similar changes in TC secretion rate. There was a linear relationship between these 2 parameters over the range 6–10 mEq Ca/liter. The sensitivity of the assay was such that TC secretion could not be detected with certainty at calcium concentrations below 6 mEq/l, although indirect evidence from other experiments indicated that it does occur. The TC secretion rate was usually small relative to the total TC content of the thyroid gland. (Endocrinology 83: 161, 1968)

Rat kidney homogenate corresponding to 50–100 mg wet tissue inactivated the calcium mobilizing activity of 40 USP U of Parathyroid Injection (Lilly) upon incubation in Robinson's buffer at 38 C for 1 hr more effectively than the liver, spleen and muscle homogenates of similar concentration. The parathyroid hormone (PTH)-inactivating activity was mainly found in the protein fraction of the kidney homogenate precipitated at 30–60% ammonium sulfate saturation and labile to heating at 65 C for 10 min. The PTH-inactivating effect was absent in the supernatant of the 0.25M sucrose homogenate of the kidney but present in the supernatant of the distilled water homogenate, suggesting the localization in the particulate fraction. PTH-inactivating effect was inhibited by mercaptoethanol at 4×10-1M, carbobenzoxy-Ltyrosin-p-nitrophenylester at 6 × 10-2M, tosyl-Larginine- o-methylester at 4×10-2M, and benzoyldl- arginine naphthylamide at 3×10-2M. EDTA inhibited PTH-inactivating effect at 1×10-2M but puromycin appeared ...

The influence of Ca++, Mg++, cysteine, 2,4-dinitrophenol, a trypsin inhibitor (Trasylol) and an antiplasmin drug (epsilon-aminocaproic acid) on the inactivation of parathyroid extract (PTH) was studied, using aerobic incubation with rat kidney slices in Robinson's buffer, pH 7.4, at 37 C for 1 hr, followed by use of the Munson assay for calcium-mobilizing activity. Calcium ions in a concentration of 12.5 mM/l, the trypsin inhibitor and epsilon-aminocaproic acid markedly inhibited the inactivation of parathyroid hormone by rat kidney slices, while 11.0 mM/1 of magnesium ions, cysteine and 2,4- dinitrophenol had no such effect. The incubation medium itself, obtained after preincubation with rat kidney slices, scarcely inactivated the PTH activity, indicating that the site of inactivation was within the slices. Kidney slices obtained from rats injected with 3.0 g/kg of sodium tartrate subcutaneously were devoid of PTH-inactivating ability, while those obtained from animals to which 0.05% HgCl2 was given by r...

The inhibitory effect of estrogens on the development of immobilization osteoporosis in the rat was studied through the analysis of calcium and hydroxyproline content of the femur and tibiae, and the measurement of cortical thickness of the femurs. A marked decrease in the calcium content of the right femur and hydroxyproline content of the right tibiae of rats in which the right hand limb was immobilized through the application of a plaster cast was significantly inhibited by the simultaneous oral administration of 100 μg of conjugated estrogens. Furthermore, administration of conjugated estrogens significantly protected against the decrease in the bone density and the cortical thickness of the femurs of the immobilized limbs of rats. It is concluded that conjugated estrogens diminish the effect ot immobilization in the development of osteoporosis, possibly through the inhibition of bone resorption. (Endocrinology 88: 102, 1971)

Upon subcutaneous injection immediately after parathyroidectomy, valine and tryptophan elevated plasma calcium concentrations and methionine decreased plasma calcium in rats fed on a diet containing 1.76% calcium. The hypercalcemic action of valine and tryptophan became evident 4 hr after injection. In rats with intact parathyroid glands, the hypercalcemic action of valine and tryptophan and the hypocalcemic action of methionine were not demonstrable, suggesting the importance of the parathyroids in calcium homeostasis. Thirteen other amino acids failed to show a measurable effect on plasma calcium in parathyroidectomized rats. {Endocrinology 78:1082,1966) A LTHOUGH the effects of orally adminis-t\. tered amino acids on intestinal absorption of calcium have been pointed out (1), parenteral administration of amino acids has scarcely been used in the study of calcium metabolism, especially with reference to plasma calcium levels. In the course of screening various amino acids for their effect on plasma calcium in rats, definite hypercalcemic effects of valine and tryptophan and a hypocalcemic effect of methionine were observed in parathyroidectomized rats fed on a diet containing 1.76% calcium. Materials and Methods Male Wistar rats of 150-200 g maintained on Oriental Rat Chow containing 1.76% calcium and 200 IU of vitamin D were parathyroidectomized by cautery. Seventeen amino acids: glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, arginine, histidine, lysine, threonine, methionine, phenylalanine, tryptophan, tyrosine, proline and hydroxyproline, were dissolved in distilled water, 100 mg/3 ml, with addition of 0.3 ml of 6N NaOH in case of glutamic acid, tyrosine and tryptophan, and subcutaneously injected immediately after parathyroidectomy at a dose of 100 mg/100 g body weight, while similarly parathyroidectomized controls received only distilled water by injection. All animals were sacrificed 6 hr later, and blood samples obtained from the jugular vein were placed in heparinized test tubes. Plasma was separated immediately, and the plasma calcium concentration was determined by stepwise titration with EDTA using a photoelectric colorimeter (2). Valine, tryptophan and methionine were also injected subcutaneously in intact rats, using similarly prepared samples, to determine the effects on plasma calcium 6 hr later. Blood samples were also obtained 1, 2 and 4 hr after injection of valine, Received September 29, 1965. tryptophan and methionine in parathyroidectomized animals. Fifty, 25 and 10 mg of valine, and 50 mg of tryptophan and methionine, were also tested to determine their effect on plasma calcium in parathyroidectomized rats fed on the diet containing 1.76% calcium.

The mechanism of the hypercalcemic effect of l-valine was studied in parathyroidectomized rats. Although a definite hypercalcemic effect of l-valine was demonstrated in parathyroidectomized rats fed a diet containing 1.85% calcium, this effect was almost completely abolished in parathyroidectomized rats fed a calcium-free diet, and also in rats in which the total intestinal tract was removed prior to parathyroidectomy. The hypercalcemic effect of valine was observed following removal of the distal jejunum, ileum or colon but was abolished by removal of the duodenum or proximal jejunum. These findings, together with the observation that subcutaneous injection of l-valine increased the speed of appearance of 45Ca in plasma following oral administration, suggest that the resulting hypercalcemia may be primarily due to enhanced intestinal absorption, although participation of other factors cannot be ruled out entirely. (Endocrinology 80: 200, 1967)

Rat parathyroid glands were cultured in 0.5 ml Eagle's Minimum Essential Medium, 0.5 ml heat-inactivated (56 C, 30 min) thyroparathyroidectomized rat serum, NaHCO3, penicillin and streptomycin for 6 to 96 hr at 37 C under 95% O2 and 5% CO2. Parathyroid hormone (PTH)-like substance released into the medium immunologically reactive with guinea pig antibovine PTH antiserum was assumed to represent rat PTH. This PTH was definitely detectable in the medium after 6 hr culture of 5 glands and increased as the incubation continued. More PTH was released as the number of glands was increased, while no detectable PTH was released from parathyroid glands frozen and thawed several times before culture. Release of PTH into medium increased as the concentration of calcium or magnesium in the medium was decreased. (Endocrinology 95: 30, 1974)

The inhibitory effect of endogenous estrogens on the parathyroid hormone (PTH) induced bone changes in rats was studied through the analysis of calcium and hydroxyproline content of the femur and tibia, and the measurement of cortical thickness of the femurs. Administration of 30 USP units of PTH for 3 weeks caused a significant fall in calcium content of the right femur and hydroxyproline content of the right tibia in both intact and ovariectomized rats. However, the magnitude of PTH induced bone changes was significantly greater in ovariectomized rat than in intact ones. Furthermore, decrease in cortical thickness of the femur caused by PTH was significantly greater in ovariectomized rat than in intact ones. It is concluded that the sensitivity of bone to PTH is increased in the absence of endogenous estrogens. (Endocrinology 90: 760, 1972)

The effect of increasing age in rats on the recovery from CaCl2-induced hypercalcemia, the hypocalcemic response to thyrocalcitonin (TC) released by cautery, and the response to increasing exogenous doses of TC were examined. The recovery from hypercalcemia was markedly retarded with advancing age. The maximum hypocalcemic response after cautery and after sc TC was largest, 3–5 mg/100 ml, in 38–39-day-old rats, smaller, 1.5–2.0 mg/100 ml, in 97–115-day-old rats, and smallest, about 1 mg/100 ml, in discarded breeders about 1 yr old. While the maximum response in discarded breeders could be increased from 1 to 1.5 mg/100 ml by repeated administration of TC, the maximum fall in serum calcium was still smaller than in younger rats. It is concluded that the delayed recovery from hypercalcemia and the smaller response to TC in old rats is due to a reduced rate of bone metabolism with advancing age. Evidence is also presented showing that in response to hypercalcemia TC is released in large amounts from the thyr...

Effects of electrical stimulation of the superior ganglion of the cervical sympathetic trunk and the administration of epinephrine or phentolamine on the expected time of recovery (ETR) from hypocalcemia induced by oxalate infusion were studied. The recovery was significantly faster in animals in which the superior cervical ganglion was electrically stimulated than in intact animals. Some acceleration of the recovery was also observed upon epinephrine administration, while the administration of phentolamine resulted in a retarded recovery. Effects of sympathetic stimulation and epinephrine administration were absent in thyroparathyroidectomized dogs. Sympathetic-parasympathetic antagonism in the regulation of the parathyroid function is suggested. (Endocrinology 76: 58, 1965)

The hypocalcemic action of sc porcine thyrocalcitonin (TC) administered alone or together with sodium phosphate was studied in rats. The dose of TC required to produce hypocalcemia was much lower in young male rats (27–47 days old) after an 18-hr fast than after feeding a diet with a high calcium to phosphate ratio (Ca/P = 5, Ca = 1.5%) for one week. However, the maximum 75-min response of serum calcium in the rats fed the high Ca/P diet was as great as that in the fasted rats. In older male rats (about 1 yr old), serum calcium fell 10% 2½ hr after TC alone, and 15% when phosphate (not hypocalcemic by itself) was given with TC. These effects in the older rats and in the young rats fed the high Ca/P diet seemed too large to be due solely to inhibition of the rate of bone resorption, which, in both situations, probably was lower than in the young fasted rats. Instead, the results suggested that TC, both alone and with phosphate, must cause hypocalcemia at least in part by another mechanism. In other studies...

The role of inorganic pyrophosphatase in the mechanism of action of parathyroid hormone and calcitonin was studied by measuiing the enzyme activity of the tibia of rats following the administration of PTH and/or calcitonin. Calcitonin caused a significant and rapid increase in the pyrophosphatase activity of the tibia which preceded the development of significant hypocalcemia. PTH, on the other hand, significantly decreased the enzyme activity of the tibia in thyroparathyroidectomized rats. These results suggest that the alteration of pyrophosphatase activity is directly involved in the mechanism of action of calcitonin and PTH. In view of the fact that pyrophosphate plays a key role in the regulation of bone formation through the inhibition of calcification, as has been suggested by Fleisch et al., its removal by pyrophosphatase would facilitate bone mineralization. (Endocrinology 89: 852,1971)

Parathyroid extract was incubated aerobically with slices of rat kidney, liver, spleen and skeletal muscle and with rat plasma in phosphate buffer, pH 7.4, at 37 C for 1 hr and the supernatant was assayed for calcium-mobilizing activity according to Munson. Slices of the kidney almost completely inactivated the extract, while other tissues were relatively ineffective. Moderate inactivation was also demonstrated upon incubation with plasma. Boiling for 15 min reduced the inactivating capacity of kidney slices. Inactivation seemed to be most effective at pH 7.4. The degree of inactivation seemed to be approximately proportional to the number of slices and the length of the incubation time. (Endocrinology 76: 255, 1965)

The oxalate tolerance test was performed for calculation of the expected time of recovery (ETR) in dogs after thyroparathyroidectomy, nephrectomy, bilateral ureteral ligation, subcutaneous injection of sodium tartrate and transureteral infusion of HgCl2. The recovery from hypocalcemia was significantly faster after nephrectomy and following proximal tubular damage by means of tartrate injection than in controls, and after ureteral ligation or distal tubular damage through retrograde HgCl2 injection into the ureter. In thyroparathyroidectomized-tartrate-injected or thyroparathyroidectomizednephrectomized dogs, the recovery was retarded, indicating the indispensability of parathyroids in the recovery from hypocalcemia. Nephrectomy and proximal tubular damage accelerated the recovery from hypocalcemia, possibly through stimulation of the parathyroid activity, while ureteral ligation and distal tubular damage had no effect. (Endocrinology 75: 857,1964)