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We investigated the in vitro effect of corticosteroids on the responsiveness of human cells of osteoblast lineage to parathyroid hormone (PTH). Prior to corticosteroid treatment, the cells demonstrated only a small increase in cAMP production and no measurable change in transmembrane potential in response to PTH. Exposure of cells to dexamethasone resulted in a 5-fold increase in PTH-induced cAMP production and in measurable PTH-induced membrane depolarization in all cells studied. The effect of corticosteroids on cAMP production was specific for PTH (not seen with PGE1 or forskolin), occurred in a time- and dose-dependent fashion and in the absence of cell proliferation. Most of the cells were of osteoblast lineage as determined by the presence of alkaline phosphatase activity and BGP secretion. These findings further support the idea that corticosteroids increase the sensitivity of cells of osteoblast lineage to PTH, perhaps by transforming cells which initially have a low responsiveness to PTH to a state of high responsiveness.

Parathyroid gland transplanted rats and hypophysectomized rats were raised from weaning on a diet without vitamin D and low in calcium (0.02%) for 4 weeks. At the enn of this period the animals of both experimental groups, when compared to their respective controls (i.e., sham-operated animals for parathyroid-transplanted ones, and hypophysectomized plus bovine growth hormone-supplemented ones for hypophysectomized rats) were characterized by (a) moderate or absent secondary hyperparathyroidism; (b) near normal bone calcium content; and (c) a maintained responsiveness to the calcemic effect of parathyroid extract (PTE). The PTE action is a bone effect that does not require the presence of the kidneys and is not related to changes in serum calcium and/or phosphorus concentrations. These results indicate that when severe hyperparathyroidism is prevented, the sensitivity of bone to the calcemic action of PTE can be maintained in D-deficient calcium-deprived rats. They also suggest that in these animals the main factor leading to resistance to PTH is the state of severe chronic hyperparathyroidism.

The endocrine response of porcine parathyroid gland tissue slices in vitro to aluminum was studied by electron microscopy and radioimmunoassay of PTH. Medium aluminum concentrations were 20 to 500 ng/ml covering the range corresponding to concentrations reported in the plasma of aluminum-intoxicated hemodialyzed patients. Aluminum inhibited iPTH-release and caused severe cell alterations. This inhibition was incomplete and there was an aluminum-insensitive iPTH-release capacity. This phenomenon seemed to be due to heterogeneous parathyroid cell population as regards aluminum sensitivity, perhaps linked to the spontaneous asynchronous cyclic parathyroid cell changes. Sensitivity to aluminum was modulated by the extra-cellular calcium concentration. Sensitivity to extra-cellular calcium concentration variations persisted in aluminum intoxicated tissues. The severity of the observed cell lesions induced by high concentrations of aluminum suggested that the recovery of an iPTH-release capacity when parathyroid tissue was withdrawn from a toxic environment and switched to aluminum-free media is more likely to be due to activation of a "less-sensitive to aluminum" cell pool than to a true reversibility of the toxic effect.

The introduction of parathyroid hormone [bPTH (1-34)], 10(-8) M, into the medium of cultured rat osteoblasts results in rapid (less than 1 min) depolarization of the osteoblast membranes. Conventional and pH-sensitive microelectrodes were used to assess the mechanism underlying this change. PTH depolarized cell membrane independently of steady-state membrane potential (Vm). Blocking K+ conductance (Ba2+) and Ca2+-dependent K+ conductance (quinine) depolarized Vm by +13.1 +/- 4.6 (n = 6) and +14.8 +/- 6.7 mV (n = 6), respectively, and both abolished the effect of PTH on Vm. The rate of depolarization was reduced in low-Ca2+ medium. PTH inhibited low Na+-induced cell hyperpolarization, but intracellular pH was not altered by hormone addition. PTH-induced depolarization occurred even when the Na+-K+ pump was blocked with ouabain. A second slower response was seen in cells having a Vm lower than -60 mV, with an increase in negativity 5-15 min after hormone application. The results indicate that PTH rapidly modifies Vm by changes of K+ conductance, which may be the first step in hormonal stimulus-response coupling, and induces delayed, long-term changes in cell status.

In order to evaluate the role of intrinsic defects in osteoblast function in the pathogenesis of diseases of skeletal development, we developed techniques which permit the evaluation of the metabolic properties of bone-derived cells in vitro. Cells from control children demonstrated a variety of properties classically attributed to osteoblasts (presence of alkaline phosphatase positive cells and synthesis of bone gla protein) and responded to PTH (cAMP production) and to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ([3H]25-hydroxyvitamin D3 conversion into [3H]24,25-dihydroxyvitamin D3 and bone gla protein secretion). Using these techniques we evaluated the function of cultured bone cells from patients with three rare diseases of skeletal development. Cells from a patient with rickets resistant to 1,25(OH)2D3 were resistant to 1,25(OH)2D3 but responded normally to PTH. Cells from a patient with acroosteolysis with osteoporosis responded normally to PTH and 1,25(OH)2D3. Cells from a patient with hyperphosphatasia with osteoectasia responded normally to 1,25(OH)2D3 but did not respond to PTH. The results demonstrate that bone cell cultures can provide information about the role of osteoblast dysfunction in such diseases.

The in vitro effects of vitamin D3 metabolites, parathyroid extract (PTE), purified parathyroid hormone (bPTH), vitamin A, and heparin on acid and alkaline phosphatases in rat or mouse calvaria in culture were investigated. Results show that: (a) when compared to values found in half calvaria incubated for 24 h in control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with PTE (L USP/ml), bPTH (4 x 10(-8)M), heparin (5 USP/ml), vitamin A (23 USP/ml), 25-(OH)D3 (2.5 x 10(-11) to 2.5 x 10(-8)M), 24,25-(OH)2D3, and 1,25-(OH)2D3 (2.5 x 10(-12) to 2.5 x 10(-7M); (b) the presence of 24,25-(OH)2D3 at low concentrations in the incubation medium decreases significantly the PTE, bPTH, vitamin A, or heparin induced stimulation of the phosphatase activities. This interaction is also observed when measuring beta glucuronidase and glucose-6-phosphatase activities and 45Ca release from previously labeled mouse calvaria; (c) a similar activity could not be found with 1,25-(OH)2D3 suggesting that 24,25-(OH)2D3 may have a specific role in bone metabolism.

Aluminum (Al) accumulation in bone is associated with low bone formation and mineralization rates; resorption may also be reduced. The mechanism of these Al-induced changes was investigated using cultured mouse osteoblast-like (OB) and osteoclast-like (OC) cells. The Al effect on bone resorption was measured by the in vitro release of 45Ca and beta-glucuronidase from mouse fetal limb-bones. Al had a biphasic effect. High concentrations (greater than 1.5 X 10(-6) M) of Al inhibited collagen and DNA synthesis, ornithine decarboxylase and alkaline phosphatase activity in OB, and depressed tartrate-resistant acid phosphatase activity in OC. Lower Al concentrations stimulated these cellular activities and 45Ca and beta-glucuronidase release from fetal bones. Al had no effect on basal cAMP levels in OB but inhibited the stimulating effect of bPTH on cAMP content. Al also altered the 1,25(OH)2D3 effects on the ornithine decarboxylase activity of OB cells. These data suggest that: (i) the low bone formation observed in vivo during Al intoxication may be due to the inhibition of collagen synthesis and to depressed cell proliferation; and (ii) Al may indirectly influence bone remodeling by interfering with the actions of bPTH and 1,25(OH)2D3 on bone cells.

Microelectrodes were used to investigate the possible involvement of cAMP and Ca2+ ions in the parathyroid hormone's, bPTH(1-34), effect on the membrane potential of rat osteoblasts in primary culture. Parathyroid hormone (10(-7) M) depolarized cell membrane by 25.0 +/- 6.1 mV (mean +/- standard deviation, SD; n = 17). Blocking Ca2+ influx with the Ca channel blocker cobalt revealed two phases in the hormone effect: a rapid and slight membrane hyperpolarization followed by sustained depolarization. In addition, cobalt significantly (p less than 0.01) decreased the magnitude of the PTH depolarizing action. The addition of dibutyryl-cAMP (10(-3) M) to the perfusion solution also resulted in a biphasic effect. At a lower concentration (10(-4) M), dibutyryl-cAMP produced only membrane hyperpolarization, suggesting a cAMP dose dependence of the opposite membrane potential changes. Forskolin (10(-5) M) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (10(-4) M) mimicked the depolarizing effect of PTH. IBMX at a low concentration (5 x 10(-6) M) potentiated the depolarizing effect of PTH. Increases in [Ca2+]i using Ca2+ ionophore A23187 and intracellular injection of CaCl2 or inositol trisphosphate decreased the PTH depolarizing action, whereas intracellular injection of EGTA enhanced this effect. These results indicate that PTH evokes a biphasic change in rat osteoblast membrane potential that seems to be mediated by an increase in cAMP and modulated by intracellular calcium.

Deflazacort was substituted for Prednisone (based on the equivalence 1 mg Prednisone equals 1.2 mg Deflazacort), during maintenance glucocorticoid therapy in 9 children, 5 with renal diseases and 4 with connective tissue or immunoproliferative disorders. Six patients received 0.26-0.35 mg/kg body weight (B.W.)/day and 3 0.48-1.2 mg/kg B.W. on alternate days, for 10-16 months. Except for a child with chronic juvenile arthritis, who was also unresponsive to Prednisone, the therapeutic effects of Deflazacort were excellent. Steroid side effects present in 8 patients decreased or disappeared. Plasma Ca, P, Mg, creatinine, alkaline phosphatase, iPTH(1-34), urinary excretion of Ca, cAMP, and TRP remained normal. Plasma iPTH(1-84) remained normal in 5 children; in the other 4 patients it increased from normal to slightly elevated values. On Deflazacort, plasma calcidiol concentrations were within the normal range in 6/8 patients prescribed daily doses of vitamin D2 (1,600-2,400 IU) or calcidiol (20 micrograms). Plasma 1,25(OH)2D levels monitored in 5 children were also normal. The osteoporosis, evaluated on the tibial cortico-diaphyseal ratio and the trabecular aspect of bone radiograms, present in 5 patients, persisted in 1 and improved in the others. On Deflazacort, statural growth proceeded normally in all subjects, with a modest acceleration of growth velocity in 3 children. These results seem encouraging for extending clinical trials with Deflazacort to the active phase of pediatric diseases requiring glucocorticoid. [ABSTRACT FROM AUTHOR]

Gallium nitrate (GaN) reduces cancer-related hypercalcemia and inhibits bone resorption in vitro. This study investigated the effects of chronic GaN administration on bone, kidney, and parathyroid gland activity of growing rats. Experimental animals received GaN (1.75 mg elemental gallium i.p. QOD X 8, Ga+), and controls received the solvent (Ga-). In the bone of Ga+ rats the number of osteoclasts was increased (Ga+: 70.4 +/- 2.31 osteoclasts/mm2; Ga-: 46.5 +/- 1.61 osteoclasts/mm2, P less than 0.001), and apposition rate and osteoid width were unchanged. Ga was concentrated in bone (2.4 mumol/g cortical bone) and detected by electron microprobe on the surface of a few trabeculae. Alkaline (Alp) and acid (Acp) phosphatase activities were higher in Ga+ than in Ga- calvaria (Ga+: Alp 223 +/- 23.4 U/mg prot, Ga-: Alp 145 +/- 13.3 U/mg prot, P less than 0.02; Ga+: Acp 69.5 +/- 4.7 U/mg prot, Ga-: 57.5 +/- 2.8 U/mg prot, P less than 0.05). Serum iPTH was increased (Ga+: 112.9 +/- 17.6 pg/ml, Ga-: 41.4 +/- 7.4 pg/ml, P less than 0.01), serum calcium was reduced (Ga+: 2.4 +/- 0.02 mmol/l, Ga-: 2.6 +/- 0.03 mmol/l, P less than 0.001); calciuria remained comparable to controls. Relative to the hypocalcemia this suggests renal loss of Ca. The calcemic response to hPTH 1-34 (i.v. 50 micrograms/kg) was decreased 2 hours after injection of the hormone (delta Ca: TPTX Ga+: 0.11 +/- 0.04 mmol/l, Ga-: 0.33 +/- 0.03 mmol/l P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) [ABSTRACT FROM AUTHOR]