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.

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.

Demonstration of sodium/calcium exchange in rodent osteoblasts.

Based on the inhibition of stimulated Ca release from cultured bone by several different agents that alter Na transport, we proposed that hormonally stimulated bone resorption requires Na/Ca exchange. Calcemic hormones appear to interact primarily directly with the osteoblast, which then mediates the activation of osteoclast activity. In organ culture it is not possible to determine whether Na/Ca exchange is involved in this initiating step in the osteoblast or directly in osteoclast-mediated Ca release, and there have been no prior direct measurements of Na/Ca exchange in bone or bone cells. The purpose of this study was to demonstrate the presence of Na/Ca exchange transport in the osteoblast. Thus, we characterized Na-dependent Ca transport in osteoblast-like rat osteosarcoma cells (UMR-106) and primary bone cells isolated from neonatal mouse calvaria. Cells were loaded with the Ca-sensitive dye fura-2 in the presence of physiologic NaCl and the absence of Ca with or without 0.3 mM ouabain. Changes in free cytosolic Ca after the extracellular addition of 1.5 mM Ca were measured spectrofluorimetrically. An outward Na gradient was generated by decreasing extracellular Na while maintaining isotonicity. UMR-106 cells that were Na loaded by ouabain inhibition of Na,K-ATPase activity exhibited 30% greater Ca uptake than control cells. Similar results were obtained with primary bone cells. This uptake required extracellular Ca, was not inhibited by 200 microM verapamil, and was reversible upon reversal of the Na gradient. These data demonstrate the presence of a Na/Ca exchange transport system in osteoblasts.

Inhibition of bone resorption by alpha-difluoromethylornithine may not be mediated by polyamine depletion.

We have examined the effect of alpha-difluoromethylornithine (DFMO) on bone polyamine content and parathyroid hormone (PTH)- and calcitriol-stimulated bone resorption in cultures of neonatal mouse calvaria. Polyamine content in bone homogenates was determined by reverse-phase paired-ion HPLC. Treatment with 5 mM DFMO for 48 h reduced putrescine from 0.4 nmol/bone to nondetectable levels, slightly decreased spermidine, and did not affect spermine. Bone resorption elicited by 48 h of treatment with PTH or calcitriol was inhibited by concentrations of DFMO greater than or equal to 5 mM added 48 h prior to hormone. This observation supported the concept that polyamines may play a role in bone resorption. However, other observations cast uncertainty on this conclusion. Measurement of calvarial polyamine content at 2 h intervals revealed no increase in endogenous polyamines for up to 10.5 h after calcitriol addition. Although addition of putrescine restored bone polyamine content, exogenous polyamines failed to reverse the inhibitory effects of DFMO on calcitriol-stimulated resorption. These results suggest that a mechanism other than depletion of polyamines could be contributing to the inhibitory effect of DFMO on resorption.

Characterization of specific thyroid hormone receptors in bone.

Thyroid hormones stimulate bone turnover in vivo and increase Ca release from bone in vitro. To investigate further the effects of thyroid hormones in bone, we have characterized specific nuclear receptors for [125I]tri-iodothyronine (T3) in neonatal mouse calvaria. Maximal specific binding of [125I]T3 to isolated nuclei occurred within 60 min at 22 degrees C. [125I]T3 binding was completely and rapidly displaced by the addition of 10(-6) M unlabeled T3; the dissociation appears to be first order with t1/2 = 36 min. The IC50 for competition by unlabeled T3 was approximately 10(-8) M. The relative affinity of thyroxine (T4) for the receptor was approximately 10 X lower than T3, consistent with its lower biological potency in most target tissues for thyroid hormones. Only weak competition was observed with diiodotyrosine at concentrations up to 10(-4) M. We have previously shown that the cardiotonic agent milrinone stimulates bone resorption in vitro with characteristics similar to those of T4. Structural homology between milrinone and T4 was recently reported. Milrinone, like diiodotyrosine, was only a weak competitor for binding at concentrations up to 10(-4) M. Milrinone inhibited collagen synthesis in the calvaria. The results suggest that the effects of milrinone on bone turnover in calvaria in vitro are probably not mediated through a thyroid hormone receptor.

Conditioned medium from ras oncogene-transformed NIH 3T3 cells induces bone resorption in vitro.

Tumor-associated hypercalcemia is due, in part, to enhanced osteoclastic bone resorption induced by soluble factors elaborated from malignant cells. ras transformation of NIH 3T3 cells results in a 50-fold induction of cathepsin L mRNA and secretion of the corresponding protein. Since cathepsin L is an acid proteinase we asked whether conditioned medium from these cells would directly increase calcium release from bone in vitro. We tested conditioned medium obtained after 72 h culture of NIH 3T3 ras-transformed cells (DT) or nontransformed NIH 3T3 cells (3T3) and identical medium not exposed to cells (Ctl). Incubation of either live or dead neonatal mouse calvaria for 48 h in DT-conditioned medium increased calcium release compared to bones incubated with 3T3 medium. In both states the increased calcium release with DT medium was blocked by 0.25 mM E-64, a general cysteine proteinase inhibitor, and 1 microM Z-Phe-Ala-CH2F, a specific inhibitor of cathepsin L activity. Thus, conditioned medium from ras-transformed cells enhances calcium release in both live and dead bone. Since cathepsin L is the major protein secreted by these cells and the effect of DT-conditioned medium is blocked by a specific inhibitor of cathepsin L, these studies suggest that this acid proteinase acts directly on bone mineral to enhance net calcium release.

Mechanism of amphotericin B stimulation of net calcium efflux from neonatal mouse calvariae.

Amphotericin B is a polyene antifungal agent that binds to membrane sterols, creating aqueous pores that permit ion fluxes sufficient to cause cell lysis. It has also been shown to alter ion transport in mammalian cells, including proton secretion from renal tubular cells. The latter effect can lead to distal renal tubular acidosis in patients treated for systemic fungal infections. Based on the understanding that osteoclast-mediated bone resorption is dependent on proton secretion, we examined the effect of amphotericin B on calcium efflux from neonatal mouse calvariae in organ culture. Amphotericin B (5 micrograms/ml) stimulated net calcium efflux from calvariae within 24 h to a level almost as great as that produced by a maximally effective concentration of parathyroid hormone. The stimulated calcium efflux was completely inhibited by both 10 ng/ml salmon calcitonin, a physiologic inhibitor of osteoclast activity, and 4 x 10(-4) M acetazolamide, a specific inhibitor of carbonic anhydrase, the enzyme necessary for substantial proton generation by osteoclasts. These results indicate a direct effect of amphotericin B on bone in vitro to stimulate osteoclast-mediated calcium efflux.

Hormonal regulation of Na(+)-Ca2+ exchange in osteoblast-like cells.

We proposed a role for Na-Ca exchange in hormonally mediated bone resorption and recently characterized Na-dependent Ca transport in an osteoblast-like rat osteosarcoma cell line (UMR-106). To test whether calcemic agents alter Na(+)-Ca2+ exchange in osteoblasts, UMR cells were treated acutely or cultured in the absence or presence of calcemic agent for 24 h. Cells were then loaded with the Ca-sensitive dye fura-2 in the presence of 140 mM NaCl, no Ca, and the absence or presence of 0.3 mM ouabain. Cells were resuspended at 22 degrees C, and the fluorescence ratio at excitation wavelength of 340 and 380 nm was measured. An outward Na gradient was generated by removing extracellular Na and maintaining isotonicity with choline chloride. Na(+)-Ca2+ exchange was demonstrated by enhanced Ca uptake in ouabain-treated (Na-loaded) cells after the addition of 1.5 mM Ca. Acute addition of 10(-7) M PTH or 10(-6) M PGE2 had no effect on Na-dependent Ca uptake. However, 24 h treatment of cells with PTH, PGE2, or 1,25(OH)2D3 caused a dose-dependent inhibition of Na(+)-Ca2+ exchange. Using the Na-sensitive dye, SBFI, we also demonstrated that the effect was bidirectional; PTH inhibited Ca-dependent Na uptake comparably to its inhibition of Na-dependent Ca uptake. The effects of the calcemic agents were mimicked by 24 h treatment of the cells with 1 microM forskolin or 2 microM PMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Dichlorobenzamil inhibits stimulated bone resorption in vitro.

We have previously proposed that stimulated release of Ca from bone requires Na-Ca exchange. 3',4'-Dichlorobenzamil (DCB), an amiloride analog, has been shown to directly block Na-Ca exchange in cardiac tissue. To further test our hypothesis we have characterized the effect of DCB on basal and stimulated bone resorption from neonatal mouse calvaria in vitro. DCB inhibition of resorption from bones stimulated with 1 nM PTH was dose dependent. The IC50 was about 7 microM, and complete inhibition occurred at 10 microM. DCB alone inhibited basal Ca release at 10 microM. Amiloride, which is less potent as an inhibitor of Na-Ca exchange, had no effect on PTH-stimulated resorption at concentrations lower than 0.1 mM, but inhibited basal resorption at 10 microM. Stimulated Ca release was inhibited either by continuous treatment with DCB plus PTH for 72 h or by a short pretreatment with DCB alone, followed by removal of DCB before addition of PTH. At least 9-h pretreatment with DCB was necessary to block the subsequent response to PTH. The inhibitory effect of DCB pretreatment could be prevented if PTH was present together with DCB during pretreatment periods of 24 h or less. This reversibility suggests that the inhibition by DCB is not simply a toxic effect of the drug. DCB also inhibited resorption stimulated by 1,25-dihydroxyvitamin D3 or prostaglandin E2, which indicates that the effect of DCB is beyond the level of specific hormone-receptor interaction. Thus, the data are consistent with a role for Na-Ca exchange in the process of hormonally stimulated bone resorption.

Inhibition of stimulated bone resorption by vanadate.

We have previously reported that stimulated release of calcium (Ca) from bone is a sodium-dependent process. Partial support for this conclusion came from the observation that ouabain inhibited stimulated bone resorption as a result of inhibition of the Na,K-ATPase in bone. We now present additional supporting evidence from the results of experiments using vanadate, an ion known to inhibit the activity of Na,K-ATPase. Vanadate inhibited stimulated bone resorption in neonatal mouse calvaria. Inhibition occurred in a dose-dependent manner, and ortho-vanadate was 3-fold more potent than meta-vanadate. Ortho-vanadate was equally effective against several different stimulators of resorption, including PTH, prostaglandin E2, and 1,25-dihydroxycholecalciferol. PTH-stimulated bone resorption was inhibited with a Ki of about 9 microM. Stimulated Ca release was completely blocked whether vanadate was added at zero time or 24 h after the addition of a resorption-stimulating agent. Because the responses to vanadate were similar to those observed with ouabain, we conclude that vanadate is probably acting to inhibit stimulated resorption via inhibition of the Na,K-ATPase in bone.

Effects of parathyroid hormone and calcitonin on osteoclast formation in vitro.

We have investigated hormonal interactions regulating bone resorption by measuring biochemical and morphological responses of neonatal mouse calvaria in vitro to treatment with 0.1 U/ml parathyroid hormone (PTH) without or with 100 ng/ml human calcitonin (CT). Calvaria were also labeled continuously with 0.5 microCi [3H]thymidine/bone. By 96 h, PTH stimulated the release of calcium (Ca) into the medium to 240% of control, while CT alone stimulated release of Ca to 96% of control. CT in the presence of PTH transiently inhibited the release of Ca up to 24--48 h after its addition. The subsequent loss of responsiveness to CT has been described as escape. Light microscopic analysis showed that PTH treatment markedly increased the number of osteoclast profiles in parallel with the stimulation of Ca release. Ct in the presence of PTH only transiently slowed the increase in osteoclast profiles. Fewer osteoclast profiles were observed in the presence of CT alone than in the control. Autoradiography of PTH-treated bones first showed [3H]thymidine label in osteoclast nuclei at 24 h, which increased markedly by 96 h. In PTH- plus CT-treated calvaria, significantly fewer osteoclast nuclei were labeled even at 96 h. In summary, hormonally induced changes in osteoclast profiles correlated well with changes in the stimulated release of Ca from calvaria. PTH had at least two effects on cell kinetics: 1) an early effect to increase the number of osteoclasts by fusion of preexisting mononuclear osteoclast precursors, and 2) a later effect to increase the fusion of postmitotic mononuclear cells with osteoclasts. In parallel with the observation of escape, CT only transiently inhibited PTH-stimulated osteoclast formation. The transient inhibition by CT of PTH-stimulated resorption can be explained by an early effect on osteoclast precursor cell fusions.

Parathyroid hormone, prostaglandin E2, and 1,25-dihydroxyvitamin D3 decrease the level of Na+-Ca2+ exchange protein in osteoblastic cells.

We previously described Na+-Ca2+ exchange in osteoblastic rat osteosarcoma cells (UMR-106) and demonstrated that Na+-dependent Ca2+ transport was inhibited by 24-hour treatment of cells with parathyroid hormone (PTH), prostaglandin E2 (PGE2), or 1,25(OH)2D3. To determine whether this inhibition of Na+-Ca2+ exchange is at the level of exchanger protein synthesis we have examined exchanger protein levels using immunoblot analysis. UMR-106 cells were treated for 24 hours with or without PTH, PGE2, or 1,25(OH)2D3. Plasma membrane fractions (7500 g) were obtained and proteins were separated by SDS-PAGE, transferred to nylon membranes, and immunoblotted with a polyclonal antibody to the canine cardiac Na+-Ca2+ exchanger. In rat cardiac membranes, we detected 125 and 75 kD bands, similar to findings for the canine exchanger. In the osteoblastic UMR cell membranes, a specific band was detected at 90 kD that decreased 65% after treatment of cells with PTH. Inhibition by PTH was dose dependent, was maximal with 10(-7) M PTH, and required 16-24 hour treatment time. Similar inhibition was observed after a 24 hour treatment with 10(-6) M PGE2 or 10(-8) M 1,25(OH)2D3. These results demonstrate the presence of a specific protein in UMR cells that cross-reacts with antibody directed against the cardiac Na+-Ca2+ exchanger. Thus, the previously reported inhibition of Na+-Ca2+ exchange activity by calcemic agents in osteoblasts appears to be due to regulation of exchanger protein levels in these osteoblastic cells.

Differential effects of parathyroid hormone on protein phosphorylation in two osteoblastlike cell populations isolated from neonatal mouse calvaria.

To further understand the mechanism of PTH effects on bone and bone cells, we have analyzed the effect of PTH on specific protein phosphorylation in cells isolated from neonatal mouse calvaria. Four populations of cells (I-IV), isolated by sequential digestion with chromatographically purified bacterial collagenase isozymes and neutral proteinase, were cultured overnight. Alkaline phosphatase activity was greater than acid phosphatase activity in all four populations. PTH stimulated cyclic AMP production in all four populations, although the effect was greatest in populations II and III. Cultured cells were treated with PTH for up to 15 minutes. Cytosolic and membrane fractions were obtained and assayed for in vitro protein phosphorylation. No hormonal effects were found in membrane fractions. In cytosol fractions, treatment of the population II cells for 10-15 minutes with 0.1 microM PTH decreased the subsequent protein phosphorylation of an 85,000 Mr protein. In contrast, PTH treatment increased in vitro phosphorylation of both the 85,000 and 35,000 Mr proteins in population III cells. Phosphorylation of the 35,000 Mr protein was cyclic AMP-dependent. All of the phosphoproteins appeared to be phosphorylated solely on serine or threonine residues except the 85,000 Mr protein which may also contain significant amounts of phosphotyrosine. Therefore, some of the effects of PTH are cyclic AMP-mediated and other effects may be mediated through tyrosine phosphorylation. These data indicate that PTH has differential effects on in vitro protein phosphorylation in two separable populations of isolated neonatal mouse calvarial cells and support a hypothesis that multiple osteoblastlike cells exist in vivo.

Comparison of fetal rat limb bones and neonatal mouse calvaria: effects of parathyroid hormone and 1,25-dihydroxyvitamin D3.

The relative responses of fetal rat limb bones and neonatal mouse calvaria to parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) were examined in organ culture. Limb bones were cultured in dishes in BGJ + 1 mg/ml bovine serum albumin (bSA) or DMEM + 15% heat-inactivated horse serum (hS). Calvaria were either cultured in dishes with one of the above two media or in roller tubes in DMEM + hS. The order of sensitivity to PTH was: calvaria in roller tubes greater than limb bones in dishes in DMEM + hS greater than limb bones in dishes in BGJ + bSA greater than or equal to calvaria in dishes in DMEM + hS greater than calvaria in dishes in BGJ + bSA. The most sensitive system (i.e., calvaria in roller tubes) showed significant resorption in response to 10(-10)M PTH at 48 h. The least sensitive system did not respond to 3 X 10(-8)M PTH after the same length of time. The greater response in DMEM + hS compared with BGJ + bSA appeared to be due to the protein component of the medium. The order of sensitivity to 1,25(OH)2D3 was: calvaria in roller tubes = long bones in culture dishes in BGJ + bSA greater than long bones in culture dishes in DMEM + hS greater than calvaria in dishes in DMEM + hS greater than calvaria in dishes in BGJ + bSA. The most sensitive systems showed resorption in response to 10(-11)M 1,25(OH)2D3 by 72 h. The least sensitive system failed to respond to 10(-9)M 1,25(OH)2D3 after the same length of time. The nature of the protein constituent did not seem to influence the response of the limb bones to 1,25(OH)2D3. The results indicated that although the responses of the two bone systems to the calcemic hormones were qualitatively similar, media and culture conditions could markedly affect the sensitivity.

Parathyroid hormone stimulates bone resorption via a Na-Ca exchange mechanism.

Parathyroid hormone (PTH) stimulates the release of calcium from bone and is thought to initiate its action by a direct effect on the plasma membrane of bone cells. Although specific membrane receptors for PTH have not yet been identified in bone, they have been characterized in kidney, and PTH does stimulate production of cyclic AMP in bone. However, the mechanism by which PTH causes the release of Ca from bone is not understood. We have determined that several agents that alter ion transport across biological membranes inhibit PTH-stimulated bone resorption in vitro. These agents include ouabain, veratridine and certain monovalent cation ionophores, which transport monovalent cations selectively. The inhibition of bone resorption is dose dependent and reversible within the first 24 h of treatment. All these compounds would be expected to increase intracellular sodium concentration in bone cells. Also, we find that decreasing the extracellular Na+ concentration prevents PTH-stimulated resorption. Therefore, we propose that PTH acts on bone to stimulate Ca release by means of a Na-Ca exchange mechanism. Decreasing the Na gradient across the bone cell plasma membrane would prevent the Na-Ca exchange and thus inhibit the physiological response to PTH.

Interaction between amrinone and parathyroid hormone on bone in culture.

The cardiotonic agent amrinone has been postulated to directly affect Na-Ca exchange. Because stimulated bone resorption has been proposed to require Na-Ca exchange, we examined the effects of amrinone on bone. Amrinone inhibited release of Ca from neonatal mouse calvaria in organ culture stimulated by parathyroid hormone (PTH), 1,25-dihydroxyvitamin d3, or prostaglandin E2. Inhibition was dose dependent and maximal at 2 X 10(-4) M. The effect of amrinone differed from the inhibitory effects of calcitonin, ouabain, or nigericin in that 1) 6-h exposure to amrinone alone prevented the effect of subsequently added PTH; 2) amrinone was only partially effective if added after resorption was initiated by 24-h treatment with PTH; 3) coincubation with amrinone and PTH during the first 48 h of culture allowed for a response to PTH after amrinone was removed; no such protection by a stimulator occurred with ouabain or nigericin. Also submaximal concentrations of amrinone plus calcitonin, ouabain, or nigericin gave greater than additive inhibition of Ca release. Amrinone had no effect on basal bone cAMP or on the acute stimulation of cAMP by PTH. The results suggest that amrinone could have a more direct interaction with the pathway involved in stimulated bone resorption than the other inhibitors.

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.

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.