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U H Lerner

Publications and source records attributed to U H Lerner.

104 records · Page 6Linked to original sources
PubMed1988

Inhibition of bone resorption in vitro by serine-esterase inhibitors.

The effect of two synthetic serine esterase inhibitors, N-alpha-dansyl(p-guanidino)phenylalaninepiperidine hydrochloride (I 2581) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (D-Phe-Pro-Arg-CH2Cl), on bone resorption in organ cultured mouse calvaria from neonatal mice has been examined. Mineral mobilization was assessed by analyzing the release of 45Ca, stable calcium (Ca2+) and inorganic phosphate (Pi). Organic matrix degradation was studied by analyzing the release of 3H from [3H]proline-labelled bones, and by quantifying the amounts of hydroxyproline in bone after culture. It was found that I 2581, at and above 30 mumol/l, dose-dependently inhibited 45Ca release induced by thrombin, parathyroid hormone (PTH), prostaglandin E2 and 1-alpha-hydroxyvitamin D-3. I 2581 (50 mumol/l) inhibited PTH-stimulated release of 3H from [3H]proline-labelled bones, and this effect was reversible after withdrawal of I 2581. I 2581 (50 mumol/l) inhibited the release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in bones stimulated by PTH and 1-alpha-hydroxyvitamin D-3, without affecting the release of lactate dehydrogenase. In parallel, I 2581 decreased PTH and 1-alpha-hydroxyvitamin D-3 induced reduction of hydroxyproline levels in bones after culture. I 2581 (50 mumol/l) did not affect the basal release of 45Ca, Ca2+, beta-glucuronidase and beta-N-acetylglucosaminidase, nor the basal amounts of hydroxyproline in bones after culture. D-Phe-Pro-Arg-CH2Cl (100 mumol/l) significantly inhibited PTH- and PGE2-induced release of 45Ca without affecting basal release of radioactive calcium. These data indicate that activation of serine proteinase(s) may be a necessary step in the mechanism of action of several stimulators of bone resorption.

Acetylglucosaminidase↗
PubMed1988

Calcitonin-like effects of forskolin and choleratoxin on surface area and motility of isolated rabbit osteoclasts.

We have previously found that the adenylate cyclase stimulators forskolin and choleratoxin increase cyclic AMP and transiently inhibit bone resorption in cultured mouse calvaria, suggesting that the compounds, directly or indirectly, may inhibit osteoclast activity. In the present study, forskolin and choleratoxin were investigated for their direct effects on surface area and motility of isolated rabbit osteoclasts, and the effects were compared to those of calcitonin (CT). Osteoclasts were cultured on coverslips for different times in the absence or presence of the compounds. The effect on osteoclast mean area was quantified on fixed and stained osteoclasts, and in addition effects were recorded with time-lapse cinemicrography. The effects of CT (100 mU/ml) on mean area and motility were seen within minutes and were maximal after 10-60 minutes. Forskolin (10-30 mumol/liter) produced a rapid (15-60 minutes) inhibition of motility and decrease in area (contraction) of osteoclasts. Choleratoxin (1 microgram/ml) treatment also resulted in cell contraction and inhibition of motility; however, the response was not seen before 45-60 minutes. The difference in the kinetics of the osteoclast response between forskolin, CT, and choleratoxin is similar to differences in time course for the effect on cyclic AMP in calvarial bones, which we reported earlier. Although cells were incubated continuously with forskolin, choleratoxin, or CT, the effects were transient. Thus, after 7-8 h incubation with CT, 3-4 h treatment with forskolin, or 4-6 h with choleratoxin, the osteoclasts started to recover from contraction and immotility. The effect of forskolin and choleratoxin on the mean surface area of osteoclasts was dose dependent. The present study shows that forskolin and choleratoxin have a direct inhibitory action on osteoclast activity and thus provide further evidence that cyclic AMP is a mediator of the action of CT on bone resorption.

Animals↗
PubMed1988

Delayed stimulation of bone resorption in vitro by phosphodiesterase inhibitors requires the presence of adenylate cyclase stimulation.

The effect on cyclic AMP levels and bone resorption by two methylxanthine cyclic AMP phosphodiesterase (PDE) inhibitors, isobutyl-methylxanthine (IBMX) and theophylline, and two non-xanthine PDE inhibitors, Ro 20-1724 and rolipram, was studied in cultured mouse calvarial bones. Cyclic AMP accumulation in calvarial bones increased when Ro 20-1724 (0.1 mmol/l) or rolipram (30 mumol/l) was present in culture medium in 2 h incubations, and when IBMX (0.3 mmol/l) or theophylline (3 mmol/l) was present in 4 h incubations. The cyclic AMP response to PDE-inhibitors could be completely abolished by the cyclooxygenase-inhibitor indomethacin (1 mumol/l). In 120 h cultures, IBMX, theophylline, Ro 20-1724 and rolipram stimulated the release of 45Ca from calvarial bones prelabelled in vivo with 45Ca. This stimulatory effect could not be seen when the endogenous production of prostaglandins was reduced by adding indomethacin (1 mumol/l), hydrocortisone (1 mumol/l) or meclofenamic acid (1 mumol/l) to culture medium. These concentrations of indomethacin, hydrocortisone and meclofenamic acid did not reduce PTH- (10 nmol/l) or choleratoxin-stimulated (0.1 micrograms/ml) 45Ca release from mouse calvarial bones cultured for 120 h. The stimulation of 45Ca release in long-term cultures by the PDE inhibitors could be demonstrated in the presence of indomethacin, provided adenylate cyclase was stimulated by forskolin (1-10 nmol/l). The stimulatory effect of 1 alpha (OH)D3 on 45Ca release could not be potentiated by the PDE-inhibitor rolipram. These results suggest that basal adenylate cyclase activity in cultured murine calvaria is very low and that therefore PDE inhibitors are inactive unless a stimulator of adenylate cyclase is present. The adenylate cyclase stimulator may be an endogenous autacoid such as a prostaglandin.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Effects of cholera toxin on cyclic AMP accumulation and bone resorption in cultured mouse calvaria.

We have utilized the adenylate cyclase stimulator, cholera toxin, as a tool to test the role of cyclic AMP as a mediator of the effects on bone resorption by the calcium-regulating hormones, parathyroid hormone (PTH) and calcitonin. The effects on bone resorption were studied in an organ culture system using calvarial bones from newborn mice. Cyclic AMP response was assayed in calvarial bone explants and isolated osteoblasts from neonatal mouse calvaria. Cholera toxin caused a dose-dependent cAMP response in calvarial bones, seen at and above approx. 1-3 ng/ml and calculated half-maximal stimulation (EC50) at 18 ng/ml. The stimulatory effect of cholera toxin could be potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 0.2 mmol/l). Cyclic AMP accumulation in the bones was maximal after 4-6 h, and thereafter declined. However, activation of the adenylate cyclase was irreversible and the total amount (bone + medium) of cAMP produced, in the presence of IBMX (0.2 mmol/l), increased with time, for at least 48 h. In osteoblast-like cells cholera toxin (1 microgram/ml) stimulated the cellular levels of cAMP with a peak after 60-120 min, which could be potentiated with IBMX. The total cAMP accumulation indicated an irreversible response. In short-term bone organ cultures (at most, 24 h) cholera toxin, at and above 3 ng/ml, inhibited the stimulatory effect of PTH (10 nmol/l) on 45Ca release from prelabelled calvarial bones. The inhibitory effect of cholera toxin (0.1 microgram/ml) on 45Ca release was significant after 6 h and the calculated IC50 value at 24 h was 11.2 ng/ml. Cholera toxin (0.1 microgram/ml) also inhibited PTH-stimulated (10 nmol/l) release of Ca2+, inorganic phosphate (Pi), beta-glucuronidase, beta-N-acetylglucosaminidase and degradation of organic matrix (release of 3H from [3H]proline-labelled bones) in 24 h cultures. 45Ca release from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxyvitamin D3 (0.1 mumol/l) was also inhibited by cholera toxin (0.3 microgram/ml) in 24-h cultures. The inhibitory effect of cholera toxin on bone resorption was transient, and in long-term cultures (120 h) cholera toxin caused a dose-dependent, delayed stimulation of mineral mobilization (Ca2+, 45Ca, Pi), degradation of matrix and release of the lysosomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Bradykinin, a new potential mediator of inflammation-induced bone resorption. Studies of the effects on mouse calvarial bones and articular cartilage in vitro.

The effect of bradykinin and desArg9-bradykinin on bone was studied in cultures of calvarial bones taken from 6-7-day-old mice. Bradykinin, at and above a 3-nM concentration, caused a dose-dependent stimulation of bone mineral mobilization and matrix degradation. Bradykinin-stimulated resorption was inhibited by calcitonin, an increased concentration of phosphate in the culture medium, hydrocortisone, dexamethasone, indomethacin, meclofenamic acid, naproxen, and 5, 8, 11, 14-eicosatetraenoic acid. The results suggest that bradykinin stimulates osteoclast-mediated bone resorption by a process that is dependent on endogenous prostaglandin production. The stimulatory effect of bradykinin, but not of parathyroid hormone and prostaglandin E2, was potentiated by the angiotensin-converting enzyme inhibitor, BPP5a. Treatment with carboxypeptidase B did not affect the capacity of the peptide to stimulate 45Ca release. DesArg9-bradykinin (1 mumole/liter) stimulated 45Ca release to the same degree as did bradykinin. Bradykinin (3 microM) did not affect the degradation of cartilage proteoglycans, as assessed by the release of 35S-sulfate from prelabeled calf articular cartilage in organ culture. These findings suggest that generation of bradykinin in inflammatory lesions of rheumatoid arthritis and periodontitis may contribute to the bone resorptive process seen in the joints and alveolar bone; however, bradykinin does not directly activate chondrocytes into a catabolic state.

Animals↗
PubMed1987

Modifications of the mouse calvarial technique improve the responsiveness to stimulators of bone resorption.

The effect of a preincubation period, in basic medium or in medium with inhibitors of prostaglandin biosynthesis, on the response to different stimulators of bone resorption has been studied in an organ culture system using calvarial bones from neonatal mice. Bone resorption was assessed either by the release of 45Ca or by the release of 3H from [3H]-proline labeled bones. Preincubated bones were cultured for 18-24 hr in medium, with and without indomethacin, hydrocortisone, and dexamethasone, and then extensively washed before being transferred to culture medium containing different stimulators of bone resorption. Preincubation in medium containing indomethacin or corticosteroids resulted in an increased response to parathyroid hormone (PTH), prostaglandin E2 (PGE2), 1-alpha-hydroxyvitamin-D3 and thrombin as compared to the response in bones which were exposed to the stimulants directly after dissection. Preincubation in basic medium did not enhance the subsequent response to PTH. By using a preincubation period in indomethacin, the dose-response curves for the stimulatory effect of PTH and PGE2 on mineral mobilization could be sensitized as compared to the curves obtained with fresh bones. Thus, the concentration of agonists causing 50% stimulation of 45Ca release was decreased by a factor of 10. The threshold for actions of PTH and PGE2 on 45Ca release was 0.01-0.03 and 1-3 nmol/l, respectively.

Animals↗
PubMed1987

Prostaglandin E2 causes a transient inhibition of mineral mobilization, matrix degradation, and lysosomal enzyme release from mouse calvarial bones in vitro.

The effect of prostaglandin E2 (PGE2) on the kinetic of bone resorption in vitro was assessed by following the release of minerals and degradation of matrix in cultured mouse calvarial bones. PGE2 (1 and 3 mumol/liter) caused an initial inhibition of the release of 45Ca, stable calcium, and inorganic phosphate from unstimulated calvarial bones. The effect was transient and after 24 and 48 hours the release of 45Ca, stable calcium, and inorganic phosphate from PGE2-treated bones was enhanced. 0.3 mumol/liter of PGE2 stimulated the release of 45Ca after 24 hours, but at this concentration no initial inhibition was observed. The initial inhibitory effect of PGE2 (1 mumol/liter) could be further increased by three structurally different inhibitors of cyclic AMP breakdown. PGE2 (1 mumol/liter) caused not only an initial inhibition of mineral release but also an initial inhibition of matrix degradation, as assessed by the release of 3H from [3H]-proline labeled bones. In addition, PGE2 (3 mumol/liter), in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, caused a rapid (6 hours) inhibition of the release of the lysosomal enzymes beta-glucuronidase and beta-N-acetyl-glucosaminidase, without affecting the release of the cytosolic enzyme lactate dehydrogenase. Similar specific initial inhibition of lysosomal enzyme release was also seen in the presence of calcitonin and dibutyryl cyclic AMP, but not in the presence of parathyroid hormone (PTH). Neither PGE2 nor the phosphodiesterase inhibitors rolipram and Ro 20.1724, could inhibit the initial stages of PTH-induced 45Ca release. Nor did PGE2 inhibit the stimulation of radioactive calcium mobilization induced by 1 alpha (OH)-vitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1987

Effects of four bisphosphonates on bone resorption, lysosomal enzyme release, protein synthesis and mitotic activities in mouse calvarial bones in vitro.

The effects of 3-amino-1-hydroxy-propylidene-1,1-bisphosphonate (AHPrBP), 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), dichloromethylenebisphosphonate (Cl2MBP) and azacycloheptylidene-2,2-bisphosphonate (AHBP) on bone were examined in organ culture using newborn mice calvaria. AHPrBP, HEBP and Cl2MBP caused a dose-dependent inhibition of PTH-stimulated (10 nmol/l) release of 45Ca from the calvaria, at and above a concentration of 3 mumol/l, whereas AHBP only caused a slight inhibition, at and above 100 mumol/l. AHPrBP inhibited PTH-stimulated release of 3H from bones prelabelled with [3H]-proline. AHPrBP (30 mumol/l) diminished the stimulatory effect of 1 alpha(OH)vitamin D3 (10 nmol/l), prostaglandin E2 (0.1 mumol/l) and renal tumor conditioned media on 45Ca release. AHPrBP and Cl2MBP, at and above 3 mumol/l, decreased PTH-stimulated mobilization of Ca2+ and Pi and in parallel the release of beta-glucuronidase without affecting the release of lactate dehydrogenase. The inhibitory effect of AHPrBP (30 mumol/l) on PTH-induced 45Ca release was irreversible. The inhibition by AHPrBP (30 mumol/l) on spontaneous and PTH-stimulated release of 45Ca can be seen first after 24 h of culture. Similarly the inhibitory effect by HEBP (30 mumol/l) and Cl2MBP (30 mumol/l) was delayed and could be observed after 36 and 24 h of culture, respectively. PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase was reduced by AHPrBP first after 24 h of culture. AHPrBP, HEBP and Cl2MBP, at concentrations which are inhibitory on bone resorption, do not affect protein synthesis and mitotic activities in mouse calvaria. These data show that AHPrBP, HEBP and Cl2MBP inhibit bone resorption in vitro and in parallel decrease lysosomal enzyme release by a mechanism, which is not related to cytotoxicity. In addition, the delayed inhibitory effect on bone resorption and lysosomal enzyme release by all the compounds suggest that bisphosphonates inhibit bone resorption indirectly and not by a direct effect on existing osteoclasts. The delayed inhibition by bisphosphonates on bone resorption may be due to decreased recruitment of new osteoclasts as a consequence of an inhibitory action on mononuclear osteoclast precursor cells.

Animals↗
PubMed1987

Characterization of adenosine receptors in bone. Studies on the effect of adenosine analogues on cyclic AMP formation and bone resorption in cultured mouse calvaria.

The effect of different adenosine analogues on cyclic AMP (cAMP) formation and bone resorption in cultured mouse calvarial bones was investigated. 5'-N-ethylcarboxamidoadenosine (NECA), R-N6-phenylisopropyl-adenosine (PIA), N6-cyclohexyl-adenosine (CHA) and 2-chloroadenosine all stimulated cyclic AMP formation with a threshold close to 1 mumol l-1); NECA was the most potent agonist. Theophylline (10, 100 mumol l-1) inhibited the cAMP accumulation induced by NECA and 2-chloroadenosine (30 and 300 mumol l-1), dose dependently. There was no inhibition of cAMP formation by PIA and CHA in forskolin-treated bone tissue. SQ 22, 536 and 2',5'-dideoxyadenosine (100 mumol l-1) both inhibited rolipram-stimulated cAMP formation. Cyclic AMP accumulation in isolated osteoblast-like cells from neonatal mouse calvarial bones was stimulated by NECA (10 and 100 mumol l-1) and 2-chloroadenosine (100 mumol l-1). 2-chloroadenosine (10 and 30 mumol l-1), but not NECA, PIA nor CHA, caused a dose-dependent stimulation of 45Ca release in both 48- and 120-h culture. The effect of 2-chloroadenosine on 45Ca release could not be antagonized by theophylline. Neither NECA, PIA, CHA nor 2-chloroadenosine could affect PTH-stimulated 45Ca release in short term cultures (6, 24 h). By contrast, stimulation of cAMP formation by forskolin or dibutyryl cAMP caused a rapid (6 h) inhibition of PTH-stimulated bone resorption. The results demonstrate functional A2 and P-site receptors in mouse calvaria and osteoblast-like cells, but no A1-receptor was detected. These adenosine receptors regulate cAMP, but are not intimately linked to bone resorption. The calcium mobilization induced by 2-chloroadenosine appears to be unrelated to adenosine receptors.

2-Chloroadenosine↗
PubMed1986

Use of forskolin to study the relationship between cyclic AMP formation and bone resorption in vitro.

The effect of the adenylate cyclase activator forskolin on bone resorption and cyclic AMP accumulation was studied in an organ-culture system by using calvarial bones from 6-7-day-old mice. Forskolin caused a rapid and fully reversible increase of cyclic AMP, which was maximal after 20-30 min. The phosphodiesterase inhibitor rolipram (30 mumol/l), enhanced the cyclic AMP response to forskolin (50 mumol/l) from a net cyclic AMP response of 1234 +/- 154 pmol/bone to 2854 +/- 193 pmol/bone (mean +/- S.E.M., n = 4). The cyclic AMP level in bones treated with forskolin (30 mumol/l) was significantly increased after 24 h of culture. Forskolin, at and above 0.3 mumol/l, in the absence and the presence of rolipram (30 mumol/l), caused a dose-dependent cyclic AMP accumulation with an calculated EC50 (concentration producing half-maximal stimulation) value at 8.3 mumol/l. In 24 h cultures forskolin inhibited spontaneous and PTH (parathyroid hormone)-stimulated 45Ca release with calculated IC50 (concentration producing half-maximal inhibition) values at 1.6 and 0.6 mumol/l respectively. Forskolin significantly inhibited the release of 3H from [3H]proline-labelled bones stimulated by PTH (10 nmol/l). The inhibitory effect by forskolin on PTH-stimulated 45Ca release was significant already after 3 h of culture. In 24 h cultures forskolin (3 mumol/l) significantly inhibited 45Ca release also from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxycholecalciferol (0.1 mumol/l). The inhibitory effect of forskolin on spontaneous and PTH-stimulated 45Ca release was transient. A dose-dependent stimulation of basal 45Ca release was seen in 120 h cultures, at and above 3 nmol of forskolin/l, with a calculated EC50 value at 16 nmol/l. The stimulatory effect of forskolin (1 mumol/l) could be inhibited by calcitonin (0.1 unit/ml), but was insensitive to indomethacin (1 mumol/l). Forskolin increased the release of 3H from [3H]proline-labelled bones cultured for 120 h and decreased the amount of hydroxyproline in bones after culture. Forskolin inhibited PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in 24 h cultures. In 120 h cultures forskolin stimulated the basal release of minerals and lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Deletions of genes encoding calcitonin/alpha-CGRP, amylin and calcitonin receptor have given new and unexpected insights into the function of calcitonin receptors and calcitonin receptor-like receptors in bone.

It has been suggested that skeletal nerves fibers may play important roles in neuro-osteogenic interactions. This view is partly based upon information obtained from immunohistochemical studies, chemical and surgical denervation experiments and clinical observations in patients with stroke and spinal cord injury, indicating the presence of a network of nerve fibers in the skeleton and that defective signalling in skeletal nerve fibers affects remodelling of bone. This view is also supported by data showing that functional receptors for signalling molecules in skeletal nerve fibers are expressed in bone cells and that activation of these receptors leads to profound effects on bone forming osteoblasts and bone resorbing osteoclasts. Convincing evidence for a role of neuronal signalling in bone metabolism has been provided by gene deletion approaches in which it has been shown that leptin-sensitive and neuropeptide Y-sensitive receptors in hypothalamus are important for bone remodelling in mice. Recently, gene deletion experiments have shown that calcitonin gene-related peptide (CGRP), one of the neuropeptides present in skeletal nerve fibers, is an important physiological regulator of bone formation at the level of osteoblast activity. CGRP belongs to the calcitonin (CT) family of peptides also including CT, amylin and adrenomedullin, as well as the recently described intermedin and calcitonin receptor-stimulating peptide. These peptides utilize two seven transmembrane G protein-coupled receptors - the calcitonin receptor (CTR) and the calcitonin receptor- like receptor (CRLR) - which can dimerize with three different single transmembrane proteins, making up the RAMP family. Associations between RAMPs and either CTR or CRLR give rise to seven distinct, molecularly characterized, receptors for CT, CGRP, amylin and adrenomedullin. Deletions of the genes for ligands in the CT family of peptides and for one of the receptors have revealed unexpected findings that have changed our view on the role of these peptides in bone remodelling. It was anticipated that deletions of the CT/alpha-CGRP and CTR genes would lead to bone loss, since CT has been shown to inhibit bone resorption in vitro and in vivo and has been used to treat patients with excessive bone resorption. Surprisingly, it was found that CT/alpha-CGRP-/- and CTR+/- mice have increased bone mass due to increased bone formation. Mice with deletion of the amylin gene, however, exhibited bone loss due to enhanced bone resorption. Selective deletion of the alpha-CGRP gene also leads to bone loss, but due to decreased bone formation. Thus, our understanding of the role of the CT family of peptides has been changed dramatically and much more data have to be gained before we fully understand the roles these peptides have in bone biology.

Amyloid↗