Fetal Rat Limb Bones Research Articles

Cadmium accelerates bone loss in ovariectomized mice and fetal rat limb bones in culture.

Loss of bone mineral after ovariectomy was studied in mice exposed to dietary cadmium at 0.25, 5, or 50 ppm. Results show that dietary cadmium at 50 ppm increased bone mineral loss to a significantly greater extent in ovariectomized mice than in sham-operated controls. These results were obtained from two studies, one in which skeletal calcium content was determined 6 months after ovariectomy and a second in which 45Ca release from 45Ca-prelabeled bones was measured immediately after the start of dietary cadmium exposure. Furthermore, experiments with 45Ca-prelabeled fetal rat limb bones in culture demonstrated that Cd at 10 nM in the medium, a concentration estimated to be in the plasma of mice exposed to 50 ppm dietary Cd, strikingly increased bone resorption, from 27 +/- 2% (mean +/- SEM) 45Ca release in cultures with no added cadmium to 68 +/- 6% release in cultures containing cadmium (n = 4). These in vitro results indicate that cadmium may enhance bone mineral loss by a direct action on bone. Results of the in vivo studies are consistent with a significant role of cadmium in the etiology of Itai-Itai disease among postmenopausal women in Japan and may in part explain the increased risk of postmenopausal osteoporosis among women who smoke.

Enhancement of fetal rat limb bone resorption by phorbol ester (PMA) and ionophore A-23187.

The present investigation was undertaken to determine if an interaction affecting 45Ca release from prelabeled fetal rat long bones could be elicited by the Ca2+ ionophore, A-23187, and the phorbol ester, 12-myristate 13-acetate (PMA). Treatment with either A-23187 at a concentration of 0.3 microM or PMA at concentrations of 10(-6) M, 10(-7) M, and 10(-8) M produced significant 45Ca mobilization. When A-23187 and PMA were combined, enhanced 45Ca release was observed on days 1 and 2 of culture. The stimulation of calcium mobilization noted on day 1 occurred when neither ionophore nor 10(-6) M PMA treatments alone produced significant 45Ca release. On day 2, cumulative 45Ca release elicited by the combination of A-23187 plus 10(-6) M PMA was slightly more than additive (15.9% for combination treatment vs. 13.7% for the sum of the individual treatments). Moreover, when A-23187 was combined with 10(-7) M PMA on day 2, an enhancement of 45Ca release was observed which was clearly more than additive (14.5% for combination treatment vs. 8.8% for the individual treatments), suggesting the possibility of a synergistic interaction between the two agents. These results were in marked contrast to those obtained with the inactive phorbol ester analog, phorbol 13-monoacetate. No stimulation of 45Ca release was observed with 10(-6) M and 10(-7) M phorbol 13-monoacetate alone nor was enhanced 45Ca release noted when the analog was combined with 0.3 microM A-23187.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiotonic agent milrinone stimulates resorption in rodent bone organ culture.

The cardiotonic agent amrinone inhibits bone resorption in vitro. Milrinone, an amrinone analog, is a more potent cardiotonic agent with lower toxicity. In contrast to amrinone, milrinone stimulated resorption in cultures of neonatal mouse calvaria and fetal rat limb bones. Threshold doses were 0.1 microM in calvaria and 0.1 mM in limb bones; maximal stimulation occurred in calvaria at 0.1 mM. Maximal responses to milrinone and parathyroid hormone were comparable. Milrinone concentrations below 0.1 mM did not affect calvarial cyclic AMP. 0.5 microM indomethacin inhibited milrinone effects in calvaria but usually not in limb bones. 3 nM calcitonin inhibited milrinone-stimulated resorption and there was no escape from this inhibition. Structural homology between milrinone and thyroxine has been reported. We find similarities between milrinone and thyroxine actions on bone, because prostaglandin production was crucial for the effects of both agents in calvaria but not in limb bones, and neither agent exhibited escape from calcitonin inhibition.

Parathyroid hormone-like adenylate cyclase-stimulating activity from a human carcinoma is associated with bone-resorbing activity.

We found previously that a human renal carcinoma cell line derived from a hypercalcemic patient induces humoral hypercalcemia when grown as allografts in the nude mouse and secretes a protein that activates adenylate cyclase via the PTH receptor. The purpose of this study was to examine the conditioned medium of this cell line for bone-resorbing activity in vitro. Processed conditioned medium produced dose-dependent stimulation of bone resorption in cultured fetal rat limb bone explants. Two PTH antagonists were used to assess the PTH receptor dependence of this bone-resorbing activity. Neither [8Nle,18Nle,34Tyr]bovine (b) PTH-(3-34) amide nor [34Tyr]bPTH-(7-34)amide inhibited bone resorption or limb bone cAMP accumulation induced by either processed conditioned medium or equivalent concentrations of bPTH-(1-34). As an alternate means to assess whether this tumor-derived PTH-like protein had intrinsic bone-resorbing activity, the latter was measured during partial purification of PTH-like adenylate cyclase-stimulating activity (ACSA) from conditioned medium by consecutive gel filtration and reverse phase HPLC. The bone-resorbing activity in conditioned medium could not be resolved from PTH-like ACSA by these two separation techniques, indicating that the activities may be intrinsic to the same protein. These results are consistent with the view that a tumor-derived protein with PTH-like ACSA and bone-resorbing activity may be responsible for hypercalcemia in vivo.

Effects of forskolin on bone in organ culture.

The effects of forskolin, which directly activates adenylate cyclase in most systems, have been compared with the actions of parathyroid hormone and calcitonin, both of which have been suggested to utilize cAMP as a second messenger in their actions on bone. Forskolin alone stimulated calcium release from neonatal mouse calvaria and fetal rat limb bones in vitro in a dose-dependent manner. The effect was maximal at 10(-6) M in both systems. At higher concentrations forskolin completely inhibited stimulated bone resorption, although with submaximal concentrations the inhibition was only partially sustained up to 72 h. Forskolin directly stimulated cAMP release from calvaria into the medium at concentrations up to 10(-4) M. Forskolin had no effect on the interaction between parathyroid hormone and calcitonin, while calcitonin inhibited the stimulatory effect of forskolin comparably with its inhibition of parathyroid hormone-stimulated bone resorption. The results indicate that forskolin has dual effects on bone and can mimic responses of both parathyroid hormone and calcitonin in both bone culture systems. The observed response depends on the concentration of forskolin used and the length of treatment with the drug.

Parathyroid hormone and calcitonin modify inositol phospholipid metabolism in fetal rat limb bones.

Inositol-containing phospholipids are believed to be intimately involved in the first steps of cellular signalling by certain hormones and neurotransmitters. We examined whether parathyroid hormone (PTH) and calcitonin (CT), two hormones that affect bone physiology, would elicit changes in inositol-phospholipid metabolism in cultured bone. [3H]inositol readily entered into the tissue phospholipid pool in fetal rat limb bones, and incorporated into phosphatidylinositol (92.9%), phosphatidylinositol-4-P (4.5%), and phosphatidylinositol-4,5-P2 (2.6%). PTH enhanced the incorporation of inositol into PtdIns in limb bones following 2- or 24-h hormone treatments. The effect of PTH was dose dependent (EC50 of 0.3-0.4 nM) and occurred in a concentration range similar to that for hormone-stimulated bone resorption. In contrast, 24-h treatment with CT-inhibited inositol incorporation, also in a dose-dependent manner. Two-hour CT treatment had variable effects on labeling. CT inhibited the stimulatory effect of PTH at both 2 and 24 h. The effects induced by PTH and CT were specific for PtdIns and were independent of the [3H]inositol pool size. These results indicate that inositol-phospholipid turnover can be modified during the action of these hormones on bone tissue. Although the time course of hormone-stimulated inositol incorporation observed here is slower than that found in other tissues, the change in phosphatidylinositol metabolism could mediate delayed effects of PTH or CT. Alternatively, alterations induced by PTH and CT in bone cell membranes, cell populations, or in the mineralized matrix could conceivably result in secondary changes in phosphatidylinositol metabolism.

Cyclosporine A inhibits calcemic hormone-induced bone resorption in vitro.

We have investigated the in vitro effects of cyclosporine (CsA), a potent immunosuppressive agent, on bone resorption induced by calcemic hormones. CsA inhibited parathyroid hormone (PTH), prostaglandin E2, 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), and osteoclast-activating factor induced resorption of fetal rat limb bones in a dose-dependent manner. Established ongoing resorptive activity in bone was also inhibited by CsA. The CsA inhibition of bone resorption could be partially surmounted by higher concentrations of PTH and 1,25(OH)2D3. The inhibitory effects of CsA on limb bone resorption were reversible. Neither protein nor DNA synthesis were inhibited by treatment of limb bones with CsA. Thus, the inhibitory effect of this agent on bone resorption is not a cytotoxic one. These data could suggest that the induction of bone resorption by the calcemic hormones involves an immune cell derived mediator such as a lymphokine.

Indomethacin inhibits thrombin-, but not thyroxin- stimulated resorption of fetal rat limb bones

The bone-resorbing effects of thrombin and thyroxin, two agents that stimulate resorption in neonatal mouse calvaria by prostaglandin-dependent mechanisms, were examined in cultures of fetal rat limb bones. Thrombin produced maximal resorption in the limb bone cultures at a concentration of 100 U/ml when bones were cultured in BGJ supplemented with 1 mg/ml bovine serum albumin. The effects of thrombin were partially inhibited by 0.5 and 10 uM indomethacin. Thrombin failed to elicit resorption when the limb bones were cultured in DMEM with 15% horse serum. Thyroxin stimulated the resorption of limb bones in both BGJ-albumin and DMEM-serum media. Resorption was elicited by thyroxin concentrations of 10 nM − 10 uM. 30 uM thyroxin failed to stimulate resorption. The dose-response curve to thyroxin was shallow, and the agent did not produce maximal resorption. The bone-resorbing effects of thyroxin were not affected by 0.5 or 10uM indomethacin.

Human transforming growth factor-alpha stimulates bone resorption in vitro.

Tumor-derived transforming growth factors (TGF) have been proposed as possible mediators of hypercalcemia in malignancy. We have studied the action of recombinant human TGF-alpha in cultured bone cells and in bone explant cultures. In clonal UMR-106 rat osteosarcoma cells, TGF-alpha and epidermal growth factor (EGF) were equipotent in binding to the EGF receptor. TGF-alpha and EGF both stimulated resorption of neonatal mouse calvaria, and maximal responses were obtained with 10 ng/ml of TGF-alpha after 72 h in culture. The effects of both TGF-alpha and EGF in calvaria, but not those of parathyroid hormone, were inhibited by 5 X 10(-7) M indomethacin. Fetal rat limb bone cultures were less sensitive to TGF-alpha than neonatal mouse calvaria, with a concentration of 30 ng/ml being required to stimulate resorption in this system. The bone-resorbing activity of TGF-alpha in fetal rat bones was inhibited by 10 ng/ml calcitonin but not by 5 X 10(-7) M indomethacin. EGF at concentrations up to 300 ng/ml did not stimulate resorption of the limb bones at time periods up to 66 h. The results indicate that human TGF-alpha is a potent bone-resorbing agent, and support the concept that this growth factor exhibits some effects distinct from those of EGF. TGF-alpha could play an etiologic role in the hypercalcemia of malignancy.

Biphasic Effects of Manganese on Hormone-Stimulated Bone Resorption*

Manganese, at concentrations of greater than 0.3 mM, inhibited PTH-stimulated resorption of fetal rat limb bones. At lower concentrations, 0.01 to 0.3 mM, manganese stimulated resorption. The bone resorbing effects of manganese were additive with those of PTH, prostaglandin E2, 1,25-dihydroxyvitamin D3, or isobutylmethylxanthine, but were potentiated by ionophore A23187. The stimulatory effects of manganese were inhibited by calcitonin but not by indomethacin. Manganese appeared to act intracellularly to stimulate resorption, in that the enhancing effects were also seen when the manganese was encapsulated in liposomes. Both stimulatory and inhibitory manganese concentrations increased cAMP in the culture medium. Manganese concentrations that inhibited PTH-stimulated resorption also inhibited the effect of PTH on medium cAMP. The enhancement of PTH-induced resorption by manganese was, however, not accompanied by increases in cAMP in the culture medium. This suggests that the PTH-Mn interaction is not mediated through the adenylate cyclase system.

10-Keto or 25-hydroxy substitution confer equivalent in vitro bone-resorbing activity to vitamin D 3

The biological activities of 10-keto derivatives of vitamin D 3 and 25-hydroxyvitamin D 3 were determined in bone organ culture. Fetal rat limb bones prelabeled with 45Ca were incubated for 60 h with 10-keto-25-hydroxyvitamin D 3, 10-keto-vitamin D 3, 1,25-dihydroxyvitamin D 3, 25-hydroxyvitamin D 3, or vitamin D 3. Resorption was quantified by release of 45Ca. Substitution of a keto group in the 10 position of the vitamin D 3 molecule resulted in a compound equal in potency to 25-hydroxyvitamin D 3. When a 10-keto group was substituted in the 25-hydroxy vitamin D 3 molecule, the potency was increased 20- to 40-fold. In contrast, 1,25-dihydroxyvitamin D 3 was 7500-fold more potent than 25-hydroxyvitamin D 3. Since 10-keto-25-hydroxyvitamin D 3 has a retention time close to that of 1,25-dihydroxyvitamin D 3 on normal-phase HPLC eluted with isopropanol:hexane, it is a possible artifact in the assay of 1,25-dihydroxy-vitamin D 3. Based upon the observed relative activities of the two compounds, the concentration of 10-keto-25-hydroxyvitamin D 3 would have to be greater than 0.8 ng/ml for it to interfere in the bioassay of 1,25-dihydroxyvitamin D 3.

24-Oxo and 26,23-lactone metabolites of 1,25-dihydroxyvitamin D 3 have direct bone-resorbing activity

The biological activities of several 24-oxo and 26,23-lactone metabolites of vitamin D were determined in bone organ cultures. The 24-oxo metabolites were significantly more potent bone-resorbing agents than the lactones. 1,25-(OH) 2-24-oxo-D 3 had 0.18× the bone-resorbing activity of 1,25-(OH) 2D 3 in fetal rat limb bones and was equipotent with 1,25-(OH) 2D 3 in neonatal mouse calvaria. In the limb bone system, 1,23,25-(OH) 3-24-oxo-D 3 had 0.08× the activity of 1,25-(OH) 2D 3. 1,25-(OH) 2D 3 and 1,25-(OH) 2-24-oxo-D 3 had a similar time course of bone-resorbing effects in both bone culture systems. The most potent of the lactones, 1,25 S-(OH) 2D 3-26,23 R-lactone, had approximately 0.009× the activity of 1,25-(OH) 2D 3 and approximately 500 times the activity of the 25 S-OH-D 3-26,23 R-lactone. The 25 S and 1,25 S lactones were more potent than the 25 R and 1,25 R isomers. In experiments designed to determine whether either 1,25-(OH) 2-24-oxo-D 3 or 25 R-OH-D 3-26,23 S-lactone could prevent the bone-resorbing activity of 1,25-(OH) 2D 3, no inhibitory effects were observed. The results suggest that conversion to the lactones represents a substantial inactivation step, whereas conversion to 24-oxo-derivatives results in less reduction in biological activity.

Potassium effects on bone: comparison of two model systems.

Effects of elevated potassium on bone resorption and on the inhibition by ouabain of parathyroid hormone (PTH)-stimulated resorption were studied in neonatal mouse calvaria, fetal mouse limb bones, and fetal rat limb bones. Ouabain inhibited PTH-stimulated resorption, and K at least partially reversed the inhibition by ouabain in all three systems. However, in contrast to calvaria, neither limb bone system was stimulated to resorb by increased K. Although the reversal of ouabain inhibition in all three systems was likely mediated by an effect on Na-K-ATPase, the resorptive effect of K alone must occur by a different mechanism because it was seen only in the calvaria. The production of prostaglandins may play a partial role in the mechanism of the stimulation of Ca release from calvaria by K. Potassium (35 mM) stimulated production of PGE2 by calvaria but not by limb bones. Indomethacin inhibited the increase in PGE2 in calvaria and partially blocked the stimulated bone resorption observed in response to K. The fetal rat limb bone cultures also differed from the mouse calvaria in being more readily inhibited by increased osmolarity. Thus, secondary effects may be responsible for the variant responses of different bone tissues to certain stimuli.

Responsiveness of vitamin D-deficient fetal rat limb bones to parathyroid hormone in culture.

Radii and ulnae from 19-day fetal rats from normal or vitamin D-deficient mothers were treated with 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, or parathyroid hormone in vitro. Both sets of bones resorbed in response to all three agents. Statistical analysis indicated a purely additive model for the effects of vitamin D status and the bone-resorbing agents, with no evidence for interaction. The results suggest that the impaired calcemic response to parathyroid hormone seen in vitamin D-deficient animals in vivo is not the result of a specific unresponsiveness of vitamin D-deficient bone to parathyroid hormone.

Ionophore A23187 promotes osteoclast formation in bone organ culture.

Morphologic effects of the ionophore A23187 were compared with those of PTH and 1,25(OH)2 vitamin D3 on fetal rat limb bone cultures. All three treatments resulted in loss of bone and the appearance of multinucleate osteoclasts. Only PTH caused proliferation of fibroblast-like cells within the marrow cavity. Cleanly dissected ("stripped") bones were unresponsive to A23187 and exhibited many dead and dying cells after 48 h in culture with this agent; in contrast, stripped bones were responsive to PTH. All three treatments showed a similar time course of appearance of osteoclasts. The results indicate that all three agents promote osteoclast formation. There are several possible explanations for the differential effects of A23187 and PTH in stripped bones, including the possibility that the cells of the periosteum are required for the action of A23187 but not of PTH.

Inhibition by cholera toxin of parathyroid hormone-induced calcium release from bone in culture

Cholera toxin was added to the culture of fetal rat limb bone and its effect on calcium release as well as on adenylate cyclase activity was examined. Cholera toxin increased the content of adenosine 3′:5′-monophosphate (cAMP) in bone. The effect on cAMP was of slower onset and of longer duration as compared with parathyroid hormone (PTH) effect. PTH added to the tissue which had been stimulated by cholera toxin increased cAMP further but the effect was partially additive. In contrary to PTH which caused a clear calcium mobilization, cholera toxin by itself had no effect or rather inhibited the release of 45Ca from the prelabeled bone. When the toxin (0.1–1 μg/ml) was combined with PTH (10 U/ml), calcium release stimulated by PTH was completely abolished.