Hollis A. Simmons

Prostaglandin synthesis by fetal rat bone in vitro: evidence for a role of prostacyclin.

Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE2, PGF2 alpha and 6-keto-PGF1 alpha, the metabolite of prostacyclin (PGI2). Since PGI2 had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI2 stimulated release of previously incorporated 45Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10(-5) to 10(-9)M. Because of the short half life of PGI2 in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI2) which was found to be a stimulator of bone resorption at concentrations of 10(-5) to 10(-6)M. These studies suggest that endogenous PGI2 production may play a role in bone metabolism. Since vessels produce PGI2 it is possible that PGI2 release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis.

Comparison of the effects of vitamin D metabolites on collagen synthesis and resportion of fetal rat bone in organ culture

SummaryWe compared the effects of four vitamin D metabolites, 1α,25 dihydroxy vitamin D3 (1α,25(OH)2D3), 1α hydroxy vitamin D3 (1αOH D3), 25 hydroxy vitamin D3 (25 OH D3), and 24R,25 dihydroxy vitamin D (24R,25(OH)2D3) on resorption and collagen synthesis in fetal rat bone maintained in organ culture. Resorption was quantitated by measuring the release of previously incorporated45Ca from long bone shafts of 19-day fetal rats, and collagen synthesis was assessed by measuring the incorporation of3H-proline into collagenase digestible protein (CDP) in calvaria from 21-day fetal rats. All four compounds stimulated bone resorption and inhibited collagen synthesis, but 1α,25(OH)2D3 was approximately 1000 times more potent in both organ culture systems. Although the differences were small among the other three compounds, the order of potency was 1αOH D3>25 OH D3≧24R,25(OH)2D3. These results suggest that the receptor for 1α25(OH)2D3 in both bone resorbing and bone forming cells has similar affinities for several vitamin D metabolites.

Effects of Parathyroid Hormone and Cortisol on Prostaglandin Production by Neonatal rat Calvaria in Vitro

We have examined the effects of parathyroid hormone (PTH) and cortisol on the production of prostaglandins, particularly PGE2, by neonatal rat calvaria cultured in a chemically-defined medium. Although there was considerable variability, calvaria produced large amounts of PGE2 in control cultures, reaching medium concentrations of 40 to 200 nM. PGE2 release was partially inhibited by cortisol at 10 nM and markedly inhibited at 100 nM. Bovine 1-34 synthetic PTH produced an increase in PGE2 concentration which was most striking in the presence of a low concentration of cortisol (10 nM). The medium also contained large amounts of 6-keto PGF1 alpha, the metabolite of prostacyclin, which showed similar changes in response to PTH and cortisol. Thromboxane B2 concentrations were low and unaffected by these hormones. 1,25-dihydroxyvitamin D did not increase medium PGE2 concentration. Since PGE2 is a potent stimulator of bone resorption and formation, some of the effects of PTH as well as cortisol may be mediated by their ability to alter PGE2 production in skeletal tissue.

Effects of prostaglandin E3 and eicosapentaenoic acid on rat bone in organ culture

To assess the possibility that diets rich in eicosapentaenoic acid (EPA) could have adverse effects on the skeleton, we examined the resorptive response to its major project, PGE3, and the effects and metabolism of EPA itself in cultured fetal rat long bones and neonatal rat calvaria. PGE3 stimulated bone resorption with a potency similar to that of PGE2. However, EPA was a much less effective precursor for PGE3 than was arachidonic acid (AA) for PGE2. In bones cultured with complement sufficient rabbit serum, which stimulates endogenous PGE release, addition of EPA had little effect on bone resorption while AA produced a substantial increase. Bones labeled with [3H]-AA and incubated with transforming growth factor-alpha (TGF-alpha), which stimulates endogenous PGE production, produced substantial amounts of PGE2, while bones labeled with [3H]-EPA and treated similarly produced less than 1/10th as much labeled PGE3. Thus, EPA appears to be a less effective precursor for the production of bone resorbing prostanoids than AA in cultured rat bone. However, since PGE3 is a potent stimulator of bone resorption, the possibility that dietary EPA can effect the production of bone resorbing prostanoids in man requires further study.

Comparison of the effects of amino-terminal synthetic parathyroid hormone-related peptide (PTHrP) of malignancy and parathyroid hormone on resorption of cultured fetal rat long bones

SummaryWe have compared the effects of of various synthetic amino-terminal forms of human parathyroid hormone-related peptide (PTHrP) of malignancy with synthetic parathyroid hormone (PTH) on the resorptive responses of fetal rat long bones in organ culture. PTH and PTHrP increased45Ca release at concentrations of 0.1–25 nM. PTHrP (1–40) and bovine PTH (1–34) were more potent than human PTH (1–34) and PTHrP (1–34). However, the slopes of the dose-response curves and the maximal resorptive effects were similar. There was a marked decrease in the potency of amino-terminal PTHrP peptides as the length was decreased. PTHrP (1–29) and PTHrP (1–25) were inactive at 120 nM. Further comparison of bPTH (1–34) and PTHrP (1–34) showed that both could induce bone resorption after a brief (6 hours) exposure and that the response to PTHrP (1–34) was qualitatively similar to that of bPTH (1–34) with respect to enhancement by ACTH and inhibition by calcitonin and glucocorticoids. Hydroxyurea and indomethacin did not block the resorptive response to either agonist. Cyclic AMP production in response to PTHrP (1–34) and (1–40) was similar to that for bPTH (1–34) in ROS 17/2.8 cells. The cyclic AMP (cAMP) response was much smaller in fetal rat long bones and calvariae, and bPTH was more potent than PTHrP. These studies confirm that PTHrP is quantitatively similar in its effects on bone resorption to PTH and are consistent with the two agents acting on the same receptor.

Biphasic effects of nonsteroidal anti-inflammatory drugs on prostaglandin production by cultured rat calvariae

We have studied the effects on bone of three structurally dissimilar non-steroidal anti-inflammatory drugs which inhibit prostaglandin cyclo-oxygenase activity (PGH synthase); indomethacin, flurbiprofen, and piroxicam. We used cultures of half calvaria from neonatal or fetal rats to measure effects on PGE2 production, measured by radioimmunoassay. In four day neonatal rat calvaria, indomethacin inhibited PGE2 release into the medium by 80% at 10(-8) M, while flurbiprofen and piroxicam produced similar inhibition at 10(-6) M. However, at 10(-10) M, treatment with all three compounds resulted in an increase in medium PGE2 concentration of 60 to 120%. To assess the mechanism of this effect, bones were labeled with [3H]-arachidonic acid, washed and cultured in the presence or absence of piroxicam. At 10(-6) M, piroxicam inhibited production of cyclo-oxygenase products and arachidonic acid release. However, at 10(-10) M, there was a substantial increase in labeled products, particularly PGE2, despite a further decrease in arachidonic acid release. In 21 day fetal rat cultures, flurbiprofen was found to increase PGE2 release both in control cultures and cultures which had been incubated with cortisol (10(-8) M) to reduce endogenous arachidonic acid release and supplied with exogenous arachidonic acid (10(-5) M) to provide a substrate. These results indicate that three potent inhibitors of PGH synthase can, paradoxically, increase prostaglandin production at low concentrations. The effect does not appear to be due to increased arachidonic acid release, and could be due to increased PGH synthase activity.

Use of osmotic minipumps for delivery of parathyroid hormone

SummaryTo determine if osmotic minipumps can be used for the local delivery of parathyroid hormone (PTH), we examined the bone resorbing activity of PTH in minipumps, either removed and assayed in bone organ cultures or released over rat calvaria. Biological activity of PTH was maintained for up to 6 days when the hormone solution contained serum and the minipumps and tubing were siliconized and flushed with diluent prior to use. Addition of cysteine did not enhance activity.