Calcitonin messenger RNA encodes multiple polypeptides in a single precursor.

Recombinant DNA techniques were used to analyze the structure of the messenger RNA encoding a precursor of calcitonin, a small calcium-regulating hormone of 32 amino acids. Analyses of the nucleotide sequences of cloned complementary DNA's comprising the entire coding sequence of the messenger RNA revealed that calcitonin is flanked at both its amino and carboxyl termini by peptide extensions linked to the hormone by short sequences of basic amino acids. The location of glycine next to the carboxyl terminal prolinamide of calcitonin is consistent with indications that glycine is required for the enzymatic amidation of proline to the prolinamide. During cellular biosynthesis, calcitonin arises from a large precursor protein by cleavages at both amino and carboxyl terminal residues of the hormone. These findings raise questions concerning the regulation of these cleavages and the potential biological functions of the precursor extensions derived from these cleavages.

Effects of glucagon, dibutyryl cyclic 3',5'-adenosine monophosphate, and theophylline on calcitonin secretion in vitro.

The secretion of calcitonin by slices of porcine thyroid glands has been investigated. Calcitonin in the incubation medium was determined by radioimmunoassay. Secretion of calcitonin was diminished when calcium or magnesium was omitted and was increased stepwise as the concentration of calcium or magnesium in the incubation medium was increased. Calcitonin secretion was augmented substantially when either the quantity of thyroid tissue or volume of incubation medium was increased. Secretion of calcitonin was stimulated by glucagon, theophylline, and dibutyryl cyclic 3',5'-adenosine monophosphate. It is concluded that calcitonin secretion is regulated by the concentration of calcium and magnesium, that secretion may be inhibited by calcitonin or a precursor and that secretion can be stimulated by increasing the concentration of cyclic 3',5'-adenosine monophosphate in the parafollicular cells of the thyroid gland.

Evidence for abnormal regulation of circulating 1 alpha,25-dihydroxyvitamin D in patients with sarcoidosis and normal calcium metabolism.

The effects of vitamin D, 2.5 mg (100,000 U)/d for 4 d, on serum calcium, serum 25-hydroxyvitamin D (25-OHD), and serum 1 alpha,25-dihydroxyvitamin D [1 alpha,25(OH)2D] were compared in 17 normal subjects and 6 patients with sarcoidosis who had normocalcemia and no history of hypercalcemia. The diagnosis was confirmed histologically in each of them. Vitamin D increased mean serum 25-PHD from 30 +/- 4 to 99 +/- 15 ng/ml (P < 0.001) and did not change mean serum 1 alpha,25(OH)2D (32 +/- 3 vs. 29 +/- 3 pg/ml) or mean serum calcium (9.5 +/- 0.1 vs. 9.6 +/- 0.1 mg/dl) in the normal subjects. In contrast, vitamin D increased mean serum 25-OHD from 19 +/- 3 to 65 +/- 19 ng/ml (p < 0.05), increased mean serum 1 alpha,25(OH)2D threefold from 40 +/- 7 to 120 +/- 24 pg/ml, and increased mean serum calcium from 9.4 +/- 0.2 to 9.8 +/- 0.2 mg/dl (P < 0.01). There was a significant positive correlation between the serum 1 alpha,25(OH)2D and serum calcium in these individuals (r = 0.663, P < 0.01) but not in the normal subjects. The results (a) provide further evidence for abnormal regulation of circulating 1 alpha,25(OH)2D in sarcoidosis and (b) indicate that the abnormality may exist in patients with normal calcium metabolism. Thus, the defect in vitamin D metabolism in sarcoid apparently is more common than was previously recognized.

Evidence that 1,25-dihydroxyvitamin D3 inhibits the hepatic production of 25-hydroxyvitamin D in man.

Previous in vitro studies in rachitic rat liver suggested that 1,25-dihydroxyvitamin D inhibits the hepatic production of 25-hydroxyvitamin D (25-OHD). An investigation therefore was carried out in eight normal subjects to determine whether concomitant administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] would alter the response of serum 25-OHD to challenge with vitamin D. In control studies, vitamin D, 100,000 U/d for 4 d, significantly increased mean serum 25-OHD, from 26.3 +/- 2.9 to 66.7 +/- 12.6 ng/ml (P less than 0.01). In contrast, 1,25(OH)2D3, 2 micrograms/d for 4 d, completely prevented an increase in serum 25-OHD in response to the same dose of vitamin D in the same individuals (25.1 +/- 2.2 vs. 27.4 +/- 5.3 ng/ml, NS). In a post-control study in seven of the normal subjects, vitamin D again significantly increased mean serum 25-OHD, from 18.2 +/- 3.1 to 42.8 +/- 4.7 ng/ml (P less than 0.001). In each of the three studies, mean serum calcium, phosphorus, and creatinine did not change and remained within the normal range. Whereas mean urinary calcium did not change in response to vitamin D alone during the 4 d of the two control studies, it increased significantly in the study in which vitamin D and 1,25(OH)2D3 were given together. A dose-response inhibition of the response of serum 25-OHD to vitamin D by 1,25(OH)2D3 was demonstrated in two of the normal subjects. The results provide evidence that 1,25(OH)2D3 inhibits the hepatic synthesis of its precursor 25-OHD in man.

Increased calcium intake does not suppress circulating 1,25-dihydroxyvitamin D in normocalcemic patients with sarcoidosis.

Ca absorption is regulated by 1,25(OH)2D, and serum values vary inversely with Ca intake. In sarcoidosis, 1,25(OH)2D is produced by alveolar macrophages in response to gamma-interferon, and patients may develop hypercalcemia after prolonged exposure to sunlight and increased dermal production of vitamin D3. To determine if increased Ca intake suppresses serum 1,25(OH)2D in normocalcemic patients and to identify those at risk, 17 normal subjects and 11 patients were studied on a metabolic ward for two and one-half days while receiving first 400 and then 1,000 mg/d of Ca. On the low Ca intake, serum angiotensin-converting enzyme (ACE), an index of disease activity, was higher in only three of the patients than in the controls, mean serum 1,25(OH)2D was higher in the patients, and mean serum total Ca, serum Ca++, and urinary Ca were not different in the two groups. On the higher Ca intake, mean urinary Ca increased in both groups, but mean serum 1,25(OH)2D was suppressed only in the normal subjects. Thus, 1,25(OH)2D production is abnormally regulated, indicating that (a) normocalcemic patients with sarcoidosis are at risk for developing abnormal Ca metabolism, and (b) a better index of disease activity is provided by the oral Ca suppression test than by serum ACE.

Demonstration of a lack of change in serum 1 alpha,25-dihydroxyvitamin D in response to parathyroid extract in pseudohypoparathyroidism.

Studies were carried out to compare the effects of parathyroid extract (PTE) on serum and urinary calcium (Ca) and phosphorus (P), serum 25-hydroxyvitamin D (25-OHD), serum 24,25-dihydroxyvitamin D (24,25(OH)2D), serum 1 alpha,25-dihydroxyvitamin D (1 alpha,25(OH)2D), and urinary cyclic AMP in two normal subjects, two patients with hypoparathyroidism (HP) and six patients with pseudohypoparathyroidism (PHP), some of whom were on suboptimal treatment with vitamin D. Two of the patients with PHP were studied while on long-term treatment with 1 alpha,25-(OH)2D3. Before PTE, serum 1 alpha, 25(OH)2D was at the lower limit of normal in one patient and was abnormally low in the other five patients. None of these individuals was on treatment with 1 alpha,25(OH)2D3. Serum 25-OHD and 24,25(OH)2D were either increased or at the upper limit of normal in the patients given vitamin D and were normal in the other patients. PTE lowered the serum P and increased the serum 1 alpha,25(OH)2D, serum and urinary Ca, urinary P, and urinary cyclic AMP in the normal subjects and patients with HP. In individual studies, changes in serum 1 alpha,25(OH)2D and serum Ca occurred in parallel before, during, and after PTE. In contrast, PTE had very little effect in the patients with PHP. Whereas there were highly significant positive correlations between serum 1 alpha,25(OH)2D in each of the normal subjects and patients with HP, there were significant correlations in only one of the patients with PHP. An increase in serum Ca in response to PTE was observed in one of the two patients with PHP who were on long-term treatment with 1 alpha,25(OH)2D3. In these individuals, PTE produced only slight increases in serum 1 alpha,25(OH)2D. Serum 25-OHD and 24,25(OH)2D were not changed by PTE in any of the subjects or patients. The results provide evidence that hypocalcemia in HP and PHP arises in part from low circulating 1 alpha,25-(OH)2D, and indicate that the lack of change in serum 1 alpha,25(OH)2D with PTE in patients with PHP is related to impaired renal adenylate cyclase and phosphaturic responses. These and previous results support the idea that diminished renal production of 1 alpha,25(OH)2D, because of a defect in the parathyroid hormone-responsive adenylate cyclase system, may be a contributing factor in the pathogenesis of the abnormal calcium metabolism in PHP.

Demonstration that circulating 1 alpha, 25-dihydroxyvitamin D is loosely regulated in normal children.

The effects of vitamin D, 2.5 mg (100,000 U)/d for 4 d, on serum calcium, serum 25-hydroxyvitamin D (25-OHD) and serum 1 alpha, 25-dihydroxyvitamin D (1 alpha, 25(OH)2D) were compared in 24 normal adults and 12 normal children. The daily dose of vitamin D was 1,500 U/kg body wt in children weighing less than 45 kg. Vitamin D increased mean serum calcium from 9.5 +/- 0.1 to 9.8 +/- 0.1 mg/dl (P less than 0.05), increased mean serum phosphorus from 4.6 +/- 0.1 to 5.0 +/- 0.1 mg/dl (P less than 0.01), increased mean serum 25-OHD from 25 +/- 3 to 34 +/- 4 ng/ml (P less than 0.001), and increased mean serum 1 alpha, 25(OH)2D from 34 +/- 3 to 42 +/- 4 pg/ml (P less than 0.02) in children. In contrast, vitamin D increased mean serum 25-OHD from 18 +/- 2 to 39 +/- 6 ng/ml (P less than 0.001) and did not change mean serum calcium (9.4 +/- 0.1 vs. 9.5 +/- 0.1 mg/dl), mean serum phosphorus (4.0 +/- 0.1 vs. 4.1 +/- 0.1 mg/dl), or mean serum 1 alpha, 25(OH)2D (31 +/- 2 vs. 29 +/- 3 pg/ml) in adults. Mean serum 1 alpha, 25(OH)2D was significantly higher after vitamin D in children than in adults (P less than 0.02). These results provide evidence that circulating 1 alpha, 25(OH)2D is not as tightly regulated in children as it is in adults. This difference in regulation could account in part for the higher values for serum 1 alpha, 25(OH)2D observed in children.

Demonstration of reduced mitogenic and osteoinductive activities in demineralized allogeneic bone matrix from vitamin D-deficient rats.

Osteoinduction is the formation of ectopic bone that follows implantation of demineralized allogeneic bone matrix (DABM) and is believed to be secondary to the release of associated inductive factors from bone matrix. To clarify the role of vitamin D in osteoinduction, we implanted DABM from vitamin D-deficient rats (-D rats) into normal rats (+D rats). Because mitogens and osteocalcin might be involved in osteoinduction, these were measured. Mitogenic activity in extracts from mineralized allogeneic bone matrix (ABM) and DABM from both +D and -D rats was determined with an assay that utilizes monolayer cultures of embryonic chick calvarial cells. Osteocalcin in serum and DABM was measured by radioimmunoassay. DABM from -D rats did not promote osteoinduction as effectively as DABM from +D rats. Resorption of implant matrix from -D rats was diminished compared with resorption of matrix from +D rats (P less than 0.01), and the decrease was attributed to a corresponding decrease in the number of osteoclasts in the implants (P less than 0.02). Bone formation (P less than 0.01) and total implant mineralization (P less than 0.001) were significantly reduced in implants from -D rats, and the reductions corresponded with a decline in the number of osteoblasts (P less than 0.05). Mitogenic activity in DABM from +D rats was only slightly decreased as compared with activity in ABM, but DABM from -D rats contained significantly less activity (P less than 0.001). No mitogenic activity was identified in implants of DABM from either +D or -D rats 3 wk after implantation. Serum osteocalcin was significantly higher in -D as compared with +D animals. In contrast, the concentrations of osteocalcin in DABM from the two groups of animals were not significantly different from each other. These findings indicate that the diminished osteoinductive activity of DABM from -D rats results from deficiency of one or more mitogenic factors that are essential for inducing the proliferation and differentiation of bone cells at the implant site and that osteocalcin does not play a role in this regard.

Evidence that increased circulating 1 alpha, 25-dihydroxyvitamin D is the probable cause for abnormal calcium metabolism in sarcoidosis.

Mean plasma 1(alpha),25-dihydroxyvitamin D[1(alpha),25(OH)(2)D] was significantly increased and serum parathyroid hormone was suppressed in three patients with sarcoidosis and hypercalcemia. Prednisone lowered the mean plasma 1(alpha),25(OH)(2)D to normal range and corrected the hypercalcemia. To elucidate the mechanism for the increased sensitivity to vitamin D in this disorder, the effects of orally-administered vitamin D(2) were determined in seven normal subjects, four patients with sarcoidosis and normal calcium metabolism and three patients with sarcoidosis and a history of hypercalcemia who were normocalcemic when studied. Serum and urinary calcium, serum 25-hydroxyvitamin D (25-OHD), plasma 1(alpha),25(OH)(2)D and, in some studies, calcium balance were measured. Vitamin D(2), 250 mug a day for 12 d, produced little, if any, change in mean plasma 1(alpha),25(OH)(2)D and in urinary calcium in the normals and in the patients with normal calcium metabolism. In contrast, vitamin D(2) produced increases in plasma 1(alpha),25(OH)(2)D from concentrations which were within the normal range (20-55 pg/ml) to abnormal values and increased urinary calcium in two patients with abnormal calcium metabolism. In an abbreviated study in the third patient, vitamin D(2), 250 mug a day for 4 d, also increased plasma 1(alpha),25(OH)(2)D abnormally from a normal value. There was a highly significant correlation between plasma 1(alpha),25(OH)(2)D and urinary calcium. Serum 25-OHD and serum calcium remained within the normal range in all subjects and patients. These findings provide evidence that the defect in calcium metabolism in sarcoidosis probably results from impaired regulation of the production and(or) degradation of 1(alpha),25(OH)(2)D. Prednisone may act to correct the abnormal calcium metabolism by reducing circulating 1(alpha),25(OH)(2)D.

Effects of dibutyryl cyclic adenosine 3',5'-monophosphate and parathyroid extract on calcium and phosphorus metabolism in hypoparathyroidism and pseudohypoparathyroidism.

It has been proposed previously that the metabolic defect in pseudohypoparathyroidism which accounts for parathyroid hormone unresponsiveness is an absence or abnormal form of the adenyl cyclase system in kidney and presumably in bone. To determine whether there is an associated defect in the response mechanism to cyclic adenosine 3',5'-monophosphate (cyclic AMP), the effects of parathyroid extract (PTE), and dibutyryl cyclic AMP were compared in patients with either surgical hypoparathyroidism or pseudohypoparathyroidism. PTE and dibutyryl cyclic AMP both increased serum and urinary calcium, lowered the serum phosphorus, and increased urinary phosphorus in patients with hypoparathyroidism. PTE also increased urinary cyclic AMP in these patients. PTE increased serum and urinary calcium and urinary phosphorus but did not alter serum phosphorus or urinary cyclic AMP in the patients with pseudohypoparathyroidism. Dibutyryl cyclic AMP increased the serum and urinary calcium, lowered the serum phosphorus, and increased urinary phosphorus in all the patients with pseudohypoparathyroidism. The results indicate that (a) dibutyryl cyclic AMP can reproduce the effects of parathyroid hormone on calcium and phosphorus metabolism in man, (b) the response mechanism to cyclic AMP appears to be intact in pseudohypoparathyroidism, and (c) PTE apparently produces some of its characteristic effects on calcium and phosphorus metabolism in pseudohypoparathyroidism in the absence of an increase in urinary cyclic AMP.

Evidence for alteration of the vitamin D-endocrine system in obese subjects.

Serum immunoreactive parathyroid hormone (PTH) is increased in obese as compared with nonobese subjects and declines with weight loss. To determine whether alteration of the vitamin D-endocrine system occurs in obesity and whether ensuing secondary hyperparathyroidism is associated with a reduction in urinary calcium, a study was performed in 12 obese white individuals, five men and seven women, and 14 nonobese white subjects, eight men and six women, ranging in age from 20 to 35 yr. Body weight averaged 106 +/- 6 kg in the obese and 68 +/- 2 kg in the nonobese subjects (P less than 0.01). Each of them were hospitalized on a metabolic ward and were given a constant daily diet containing 400 mg of calcium and 900 mg of phosphorus. Whereas mean serum calcium, serum ionized calcium, and serum phosphorus were the same in the two groups, mean serum immunoreactive PTH (518 +/- 48 vs. 243 +/- 33 pg/ml, P less than 0.001), mean serum 1,25-dihydroxyvitamin D [1,25(OH)2D] (37 +/- 2 vs. 29 +/- 2, P less than 0.01), and mean serum Gla protein (33 +/- 2 vs. 24 +/- 2 ng/ml, P less than 0.02) were significantly higher, and mean serum 25-hydroxyvitamin D (25-OHD) (8 +/- 1 vs. 20 +/- 2 ng/ml, P less than 0.001) was significantly lower in the obese than in the nonobese men and women. Mean urinary phosphorus was the same in the two groups, whereas mean urinary calcium (115 +/- 10 vs. 166 +/- 13 mg/d, P less than 0.01) was significantly lower, and mean urinary cyclic AMP (3.18 +/- 0.43 vs. 1.84 +/- 0.25 nM/dl GF, P less than 0.01) and creatinine clearance (216 +/- 13 vs. 173 +/- 6 liter/d, P less than 0.01) were significantly higher in the obese than in the nonobese individuals. There was a significant positive correlation between percentage of ideal body weight and urinary cyclic AMP (r = 0.524, P less than 0.01) and between percentage of ideal body weight and serum immunoreactive PTH (r = 0.717, P less than 0.01) in the two groups. The results provide evidence that alteration of the vitamin D-endocrine system in obese subjects is characterized by secondary hyperparathyroidism which is associated with enhanced renal tubular reabsorption of calcium and increased circulating 1,25(OH)2D. The reduction of serum 25-OHD in them is attributed to feedback inhibition of hepatic synthesis of the precursor by the increased serum 1,25(OH)2D.

Evidence for alteration of the vitamin D-endocrine system in blacks.

As compared with values in white subjects, bone mass is known to be increased and urinary calcium to be diminished in black individuals. To evaluate the possibility that these changes are associated with alterations in the vitamin D-endocrine system, an investigation was performed in 12 black subjects, 7 men and 5 women, and 14 white subjects, 8 men and 6 women, ranging in age from 20 to 35 yr. All of them were hospitalized on a metabolic ward and were given a constant daily diet containing 400 mg of calcium, 900 mg of phosphorus, and 110 meq of sodium. Whereas mean serum calcium, ionized calcium, and phosphate were the same in the two groups, mean serum immunoreactive parathyroid hormone (350 +/- 34 vs. 225 +/- 26 pg/ml, P less than 0.01) and mean serum 1,25-dihydroxyvitamin D (1,25(OH)2D) (41 +/- 3 vs. 29 +/- 2 pg/ml, P less than 0.01) were significantly higher, and mean serum 25-hydroxy-vitamin D (25-OHD) was significantly lower in the blacks than in the whites (6 +/- 1 vs. 20 +/- 2 ng/ml, P less than 0.001). Mean urinary sodium and 24-h creatinine clearance were the same in the two groups, whereas mean urinary calcium was significantly lower (101 +/- 14 vs. 166 +/- 13 mg/d, P less than 0.01) and mean urinary cyclic AMP was significantly higher (3.11 +/- 0.47 vs. 1.84 +/- 0.25 nM/dl glomerular filtrate, P less than 0.01) in the blacks. Further, the blacks excreted an intravenous calcium load, 15 mg/kg body weight, as efficiently as the whites (49 +/- 3 vs. 53 +/- 3%, NS). Mean serum Gla protein was lower in blacks than in whites (14 +/- 2 vs. 24 +/- 3 ng/ml, P less than 0.02), and increased significantly in both groups in response to 1,25(OH)2D3, 4 micrograms/d for 4 d. There was a blunted response of urinary calcium to 1,25(OH)2D3 in the blacks, and mean serum calcium did not change. The results indicate that alteration of the vitamin D-endocrine system with enhanced renal tubular reabsorption of calcium and increased circulating 1,25(OH)2D as a result of secondary hyperparathyroidism may contribute to the increased bone mass in blacks. Their low serum 25-OHD is attributed to diminished synthesis of vitamin D in the skin because of increased pigment.

Evidence for extrarenal production of 1 alpha ,25-dihydroxyvitamin D in man.

Recent studies provide evidence for extrarenal production of 1 alpha ,25-dihydroxyvitamin D [1 alpha ,25(OH)2D]. To investigate this possibility, serum vitamin D, 25-hydroxyvitamin D (25-OHD), 24,25-dihydroxyvitamin D [24,25(OH)2D], and 1 alpha ,25(OH)2D were measured in eight adult anephric subjects. All were undergoing hemodialysis and three of them were receiving vitamin D, 50,000 or 100,000 U/d. Serum vitamin D was elevated in two of the patients given vitamin D and was abnormally low in the others. Mean serum 25-OHD was increased in patients given vitamin D (94.0 +/- 7.6 ng/ml) and was normal in the others (16.4 +/- 0.9 ng/ml, P less than 0.001). Mean serum 24,25(OH)2D was normal in patients given vitamin D (1.38 +/- 0.27 ng/ml) and was low in the others (0.25 +/- 0.08 ng/ml, P less than 0.001). Serum 24,25(OH)2D correlated significantly with serum 25-OHD (r = 0.848, P less than 0.01). Mean serum 1 alpha ,25(OH)2D determined by receptor assay was 5.8 +/- 1.9 pg/ml in patients who were not given vitamin D and was 14.1 +/- 0.6 in those who were given vitamin D (P less than 0.001). Serum 1 alpha ,25(OH)2D correlated significantly with serum 25-OHD (r = 0.911, P less than 0.01). Mean serum 1 alpha ,25(OH)2D, measured by bioassay, was 8.3 +/- 1.9 pg/ml in patients who were given vitamin D and was 15.9 +/- 2.4 pg/ml in those who were given vitamin D (P less than 0.05). There was a significant correlation between the values for serum 1 alpha ,25(OH)2D obtained with the two methods (r = 0.728, P less than 0.01). The results (a) provide evidence in man for extrarenal production of both 24,25(OH)2D and, by two independent assays, of 1 alpha , 25(OH)2D, and (b) indicate that serum values of the two dihydroxy metabolites of vitamin D in anephric subjects vary with the serum concentration of the precursor 25-OHD.

Bone and mineral metabolism in African Americans.

Important differences exist in the metabolism of bone and mineral and the vitamin D endocrine system between whites and African Americans and include rate o f skeletal remodeling, bone mass, and vitamin D metabolism. A higher bone mineral density (BMD) in African Americans is associated with a diminished incidence o f osteoporosis and fractures. Serum 17beta-estradiol and the rate of GH secretion are higher in black than in white men, but there is no racial difference in women in this regard. The mechanisms for reduced rate o f skeletal remodeling and for greater BMD in blacks are not known, but diminished rate of skeletal remodeling could be a contributing factor for greater bone mass. Reduction in serum 25-hydroxyvitamin D in blacks is attributed to increased skin pigment and to diminished dermal production of vitamin D(3) and consequent decreased hepatic synthesis o f the metabolite. There is no evidence that alteration of the vitamin D endocrine system contributes to or is responsible for racial differences in skeletal remodeling and bone mass. Black infants, however, are at risk for developing vitamin D-deficient rickets, particularly when breast-fed.

Mapping the domains of the interaction of the vitamin D receptor and steroid receptor coactivator-1.

The vitamin D receptor (VDR) binds to the vitamin D response element (VDRE) and mediates the effects of the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], on gene expression. The VDR binds to the VDRE as a heterodimeric complex with retinoid X receptor. In the present study, we have used a yeast two-hybrid system to clone complementary DNA that codes for VDR-interacting protein(s). We found that the human steroid receptor coactivator-1 (SRC-1) interacts with the VDR in a ligand-dependent manner, as demonstrated by beta-galactosidase production. The interaction of the VDR and the SRC-1 takes place at physiological concentrations of 1,25(OH)2D3. A 48.2-fold stimulation of beta-galactosidase activity was observed in the presence of 10(-10) M 1,25-(OH)2D3. In addition, a direct interaction between the ligand-activated glutathione-S-transferase-VDR and 35S-labeled SRC-1 was observed in vitro. Deletion-mutation analysis of the VDR established that the ligand-dependent activation domain (AF-2) of the VDR is required for the interaction with SRC-1. One deletion mutant, pGVDR-(1-418), bound the ligand but failed to interact with the SRC-1, whereas another deletion mutant, pGVDR-(1-423), bound the ligand and interacted with the SRC-1. We demonstrated that all the deletion mutants were expressed as analyzed by a Gal4 DNA-binding domain antibody. Deletion mutation analysis of the SRC-1 demonstrated that 27 amino acids (DPCNTNPTPMTKATPEEIKLEAQSQFT) of the SRC-1 are essential for interaction with the AF-2 motif of the VDR.

The effects of immobilization on bone histomorphometry in rats.

To determine whether immobilization acts directly on bone by alteration of mechanical loading or systemically, studies of the effects of immobilization were carried out on histomorphometry of diaphyses of tibiae and on subcutaneous implants of demineralized allogenic bone matrix of rats. The right hind leg of growing rats was denervated by severing the tibial nerve. A sham operation on the right hind leg was performed in control animals. Bone formation at the endosteal and periosteal surfaces was significantly lower in tibiae from limbs with severed nerves as compared to tibiae from the intact limbs of nerve-sectioned rats and from both limbs of sham-operated control rats. Bone formation was decreased at both 3 and 7 weeks after immobilization. The decreased formation resulted in significant reductions in cross-sectional area. At 3 weeks post denervation, the periosteal bone formation rate was lower in tibiae of intact limbs from denervated rats as compared to tibiae from intact limbs of sham-operated animals. This finding was attributed to reduced physical activity of the denervated rats. In the implants, nerve section did not alter the amount of implant matrix resorbed, the amount of bone matrix synthesized, or the amount of calcium in the implant. These findings support the hypothesis that inhibition of bone formation at the tibial diaphysis in response to immobilization resulted from altered mechanical loading and not from the production of substances acting systemically. Whereas the mean medullary area of tibiae was not altered by nerve section, it was decreased in tibiae of all groups compared to the values of basal controls, indicating that bone formation was greater than bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that calcium modulates circulating 25-hydroxyvitamin D in man.

We previously demonstrated in normal subjects that 1,25-dihydroxyvitamin D3 (1,25(OH)2D) can prevent the increase in serum 25-hydroxyvitamin D (25-OHD) which occurs in response to vitamin D. An investigation was carried out in eight normal subjects, therefore, to determine whether increases in calcium intake would alter the response of serum 25-OHD to challenge with vitamin D. In control studies, vitamin D, 100,000 U/d for 4 d, significantly increased mean serum 25-OHD from 18 +/- 3 to 42 +/- 5 ng/ml (p less than 0.001), an increment of 24 ng/ml (133%). Mean serum calcium, ionized calcium, phosphorus, creatinine, and 1,25(OH)2D did not change. In contrast, the same dose of vitamin D and calcium, 2,000 mg/d for 4 d, administered to the same eight subjects produced an increase in mean serum 25-OHD from 19 +/- 3 to 31 +/- 4 ng/ml (p less than 0.001), an increment of only 12 ng/ml (63%) and significantly less than the control (p less than 0.02). Mean serum calcium (8.8 +/- 0.1 vs. 9.2 +/- 0.1 mg/dl, p less than 0.01) and ionized calcium (4.79 +/- 0.07 vs. 4.85 +/- 0.08 mg/dl, p less than 0.05) increased significantly in response to vitamin D and calcium, mean serum phosphorus and creatinine did not change, and mean serum 1,25(OH)2D decreased significantly (37 +/- 2 vs. 31 +/- 4 pg/ml, p less than 0.02). In a postcontrol study in six of the normal subjects, vitamin D again significantly increased mean serum 25-OHD from 17 +/- 3 to 39 +/- 9 ng/ml (p less than 0.02), an increment of 22 ng/ml (129%).(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration that ethanol inhibits bone matrix synthesis and mineralization in the rat.

The effects of ethanol on bone and mineral metabolism were investigated in 3 groups of male rats. The first group received ethanol administered as 36% of caloric content in a liquid diet for 3 weeks. A second group of pair-fed animals was given the same liquid diet, except that sucrose was substituted isocalorically for ethanol. A third group of rats was fed standard laboratory chow. The ethanol-treated rats gained significantly less weight than laboratory chow-fed controls but gained the same weight as the pair-fed animals. Ethanol-treated rats had a modest but significant decrease in mean serum calcium compared to pair-fed controls (10.3 +/- 0.1 vs. 10.6 +/- 0.1 mg/dl, p less than .001). Mean serum phosphate, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and immunoreactive parathyroid hormone were the same in the 3 groups. The ethanol-treated animals showed significant decreases in mean tibial length (1.88 +/- 0.01 vs. 1.98 +/- 0.02 cm, p less than .01), mean endosteal bone formation rate (0.0006 +/- 0.0001 vs. 0.0026 +/- 0.0003 mm3/day, p less than .001) and mean periosteal bone formation rate (0.022 +/- 0.001 vs. 0.026 +/- 0.001 mm3/day, p less than .01) compared to the pair-fed controls. The ethanol-treated rats demonstrated significant decreases in mean periosteal mineralization rate (7.5 +/- 0.3 vs. 10.3 +/- 0.6 micron/day, p less than .01) and mean periosteal apposition rate (8.5 +/- 0.5 vs. 11.0 +/- 0.8 micron/day, p less than .05) and a significant increase in mean periosteal osteoid thickness (15.5 +/- 1.4 vs. 10.4 +/- 0.8 micron, p less than .01) compared to pair-fed controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of ovariectomy and 17 beta-estradiol on cortical bone histomorphometry in growing rats.

The effects of ovariectomy for four weeks and of 17 beta-estradiol for three weeks on histomorphometry of the tibial diaphysis were determined in young rats. The effects of ovariectomy on histomorphometry of subcutaneous implants of demineralized bone matrix were also examined. Groups of young female rats were either ovariectomized or sham operated. After surgery, the animals were weight matched and pair fed. Despite the same caloric intake, ovariectomized rats grew more rapidly than pair-fed, sham-operated controls but were significantly heavier at sacrifice in only one of three experiments. Ovariectomy did not change mean serum calcium, phosphate, 25-hydroxyvitamin D (25-OHD), or 1,25-dihydroxyvitamin D [1,25(OH)2D] but significantly lowered mean serum magnesium. Serum estradiol was not detectable in ovariectomized animals. 17 beta-Estradiol in ovariectomized animals significantly increased mean serum estradiol and lowered mean serum phosphate but did not change mean serum calcium, magnesium, 25-OHD, or 1,25(OH)2D, as compared to values in sham-operated controls. Bone formation rate was significantly enhanced in ovariectomized animals at both the endosteal and periosteal surfaces of the tibial diaphysis as compared to values in sham-operated controls. The increase in bone formation rate was reversed by 17 beta-estradiol at the periosteal but not endosteal surface. Ovariectomy increased the bone apposition rate, mineralization rate, and osteoid thickness of the tibial diaphysis. These increases were reversed by 17 beta-estradiol. In implants, ovariectomy increased the resorption of implant matrix and enhanced the formation of new matrix. Ovariectomy resulted in increases in forming surface and resorbing surface in the implants.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that ibuprofen antagonizes selective actions of estrogen and tamoxifen on rat bone.

Studies were performed to determine if the nonsteroidal anti-inflammatory drug ibuprofen alters bone and mineral metabolism in female rats. In experiment 1, four groups of growing rats underwent either sham operation or ovariectomy (OVX). One week later, controlled-release pellets with ibuprofen or placebo were implanted subcutaneously at the back of the neck. Following 3 weeks of treatment, rats were sacrificed and blood and bone samples were removed for serum assays and histomorphometric analysis. Body growth rate and the static cortical bone measurements made at the tibial diaphysis did not change in response to OVX. OVX, however, did increase radial bone growth, lowered serum 17beta-estradiol, reduced uterine weight, and decreased the cancellous bone area of the tibial metaphysis in the rats. Ibuprofen did not alter serum 17beta-estradiol or uterine weight but reduced radial bone growth as well as cancellous bone area of the tibial metaphysis in both sham-operated and OVX animals. In experiments 2 and 3, we tested the influence of ibuprofen on the effects of the tissue-selective estrogen agonist tamoxifen and of exogenous 17beta-estradiol in the OVX rat. Ibuprofen completely blocked the effects of tamoxifen and partially blocked the effects of 17beta-estradiol to prevent cancellous osteopenia. In contrast, ibuprofen did not influence the effects of tamoxifen and 17beta-estradiol to reduce radial bone growth. Besides the skeletal effects, ibuprofen suppressed estrogen-induced uterine growth. Our data suggest that ibuprofen blocks selective estrogen receptor-mediated activities in the rat.