0013-7227/90/1262-1076$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 2 Printed in U.S.A.
Effects of Prostaglandin F2« on Bone Formation and Resorption in Cultured Neonatal Mouse Calvariae: Role of Prostaglandin E2 Production* LAWRENCE G. RAISZt, CYNTHIA B. ALANDER, PAMELA M. FALL, AND HOLLIS A. SIMMONS Division of Endocrinology and Metabolism, University of Connecticut Health Center, Farmington, Connecticut 06032
labeling of CDP in the absence of cortisol and stimulated CDP labeling in the presence of cortisol. PGF2o increased thymidine incorporation into DNA, but the effect was smaller than that of PGE2 and was inhibited by indomethacin. These observations suggested that PGF2o might act in part by stimulating PGE2 production. By RIA, PGE2 concentrations were increased in the medium of bones treated with PGF2a, and this increase was blocked by indomethacin. By HPLC, bones prelabeled with [3H]arachidonic acid showed an increase in labeled PGE2 release, and RIA showed an increase in PGE2 after PGF2a treatment. These results indicate that PGF2a is a relatively weak agonist in bone compared to PGE2 and that some of the effects of PGF2a on bone resorption, formation, and cell replication may be mediated by an increase in endogenous PGE2 production. {Endocrinology 126: 1076-1079,1990)
ABSTRACT. Although most studies show that prostaglandin E2 (PGE2) is the most potent and effective of the prostanoids in bone, recent data in cell culture suggest that PGF2a may have unique effects, particularly on cell replication. The present study was undertaken to compare the effects of PGF 2 Q and PGE2 in cultured neonatal mouse parietal bones by simultaneous measurement of bone resorption as release of previously incorporated 46 Ca, bone formation as incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein, and DNA synthesis as incorporation of [3H]thymidine. PGF2a was less effective than PGE2 as a stimulator of bone resorption, and its effects were partially inhibited by indomethacin and markedly inhibited by glucocorticoids. In contrast, the resorptive response to PGE2 was unaffected by indomethacin and only partially inhibited by cortisol. PGF2a had little effect on bone formation, in contrast to the biphasic effect of PGE2, which inhibited
M
ANY studies in organ culture have indicated that prostaglandins of the E series (PGE) are potent stimulators of bone resorption and also have a biphasic effect on bone formation (1-3). In organ culture, PGF 2a was also found to stimulate resorption, but was less potent than PGE2. The effects of PGF2« on formation have not been extensively studied. Recently, studies in cell culture using either rat osteosarcoma cells (UMR 106) or cloned mouse cells with an osteoblastic phenotype (MC3T3-E1) have suggested that PGF 2a may be a potent mitogen in bone and that its effects could involve a separate receptor and second messenger system from that responsive to PGE 2 (4, 5).
by the incorporation of [3H]proline into collagenasedigestible (CDP) and noncollagen protein (NCP), and DNA synthesis by incorporation of [3H]thymidine in cultures of neonatal mouse parietal bones. In this system we found that PGF 2a was less potent than PGE2 and that its effects were diminished by treatment with indomethacin and cortisol. Based on these results, we examined the effect PGF 2a on PGE2 production and found an increase in medium PGE2 by RIA (iPGE2) and by analysis of [3H]PGE2 production in bones prelabeled with [3H] arachidonic acid ([3H]AA).
In the present study we have used an organ culture system in which bone resorption can be assessed by the release of previously incorporated 45Ca, bone formation
Timed mated pregnant mice (CD-I, Charles River, Wilmington, MA) were injected with 0.05 mCi 45Ca (New England Nuclear, Wilmington, DE) on the 16th day of gestation. Sevenday-old neonatal mice were killed, and the central portion of the parietal bone ( 2 x 3 mm) was dissected free of connective tissue and suture material and precultured for 18-24 h in BGJ medium (Gibco, Grand Island, NY) supplemented with 1 mg/ ml BSA (RIA grade, Sigma, St. Louis, MO) and containing 3 mM phosphate, 1 mM proline, and 100 Mg/ml freshly added ascorbic acid. The cultures were treated with PGE2, PGF2a,
Received September 8, 1989. * This work was supported by NIH Grants AR-18063 and AR-38933. A preliminary report of this work was presented at the combined meeting of the American Society for Bone and Mineral Research and the International Conferences on Calcium Regulating Hormones, Montreal, Quebec, Canada, September 1989. t To whom requests for reprints should be addressed.
Materials and Methods
1076
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EFFECTS OF cortisol (10 7 M), or indomethacin (10 6 M; Sigma). Cortisol and indomethacin were added during the preculture period. PGs and cortisol were dissolved in ethanol and added at a 0.1% final ethanol concentration, with a similar concentration in controls. PG treatment was carried out for 1-3 days, with daily medium changes. DNA synthesis was assessed by adding [3H] thymidine (5 /aCi/ml; New England Nuclear) for the last 2 h of a 24-h culture. Cold thymidine (10~5 M) was added to culture medium for these experiments. Acid-soluble [3H]thymidine was measured in trichloroacetic acid extracts of bone, and the residual bone was counted to assess DNA synthesis. In other cultures, [3H]proline (5 fiCi/m\; New England Nuclear) was added for the last 2 h of a 72-h culture, and its incorporation into CDP and NCP was measured as described previously (2). Percent collagen synthesis (PCS) was calculated after correction for the relative abundance of proline in CDP and NCP. 45 Ca was measured in the medium and trichloroacetic acid extracts of the bone to calculate percentage of 45Ca released. The medium iPGE2 concentration was measured by RIA (6). Two antibodies were used; the antibody from Dr. L. Levine of Brandeis University cross-reacted with PGF2« at 2%, while that kindly provided by Dr. N. H. L. Hunt (Canberra, Australia) cross-reacted only 0.4%. The latter was used for direct RIA of media to which PGF2«had been added. To assess PGE2 production further, two additional experiments were carried out. In one, bones were prelabeled with [3H]AA (10 j^Ci/ml) for 18 h. The bones were then washed for 2 h in fresh medium and cultured with PGF2o, for 24 h. The media were pooled from three cultures, acidified and extracted with ethyl acetate, and the products were separated on HPLC using a gradient system that separates PGF2« and PGE2by several fractions (7). HPLC profiles showed that the predominant labeled product was [3H] PGE2, as we have reported previously for rat calvariae (8). In the second experiment the media from unlabeled bones treated with PGF2« were pooled, extracted, and chromatographed on HPLC, and the PGE2 content of individual fractions was assessed by RIA. Statistical analysis was carried out by analysis of variance. Significance of differences was determined by post-hoc testing Using Bonferroni's method.
Results There was a significant increase in 45Ca release at 72 h with PGF 2a at 10"6 and 10"5 M, but not at lower concentrations (Fig. 1). In cultures treated with indomethacin the resorptive response was diminished and was significant only at 10~5 M. With cortisol the response to PGF2a was further diminished, but the small increase in 45Ca release at 10"5 M was significant (P < 0.05). PGE2 was more potent than PGF2« and produced a greater increase in 45Ca release. Stimulation of resorption was significant at 10~7 and 10~6 M (P < 0.01) and was similar in the presence or absence of indomethacin and decreased but still significant in the presence of cortisol. PGF 2a produced little change in the incorporation of proline into CDP or NCP. The only significant effects were an increase in NCP at 10~6 M and a decrease in
PGF2CT
ON BONE
Log PGF2-alpha [M]
1077
LogPGE2[M]
FIG. 1. Effect of PGF2aand PGE2on release of previously incorporated 45 Ca for neonatal mouse parietal bones at 72 h. Points are the means and vertical lines the SE for 6-36 cultures. Effects of PGF2a are significant (P < 0.01) at 10"5 and 10~6 M in the absence and at 10"6 M in the presence of indomethacin. Effects of PGE2 are significant (P < 0.01) at 10"6 and 10~7 M with or without cortisol or indomethacin.
PCS at 10"5 M (Table 1). In the presence of cortisol, which itself decreased labeling of CDP, PCS, and bone weight at 72 h, PGF2o had no significant effect. In contrast, PGE2 showed effects on bone formation in this model similar to those that we have reported previously in fetal rat calvaria (3). In the absence of cortisol, high concentrations inhibited incorporation of proline into CDP and decreased PCS. In the presence of cortisol, however, PGE2 stimulated incorporation of proline into CDP and NCP and increased bone weight. Both PGF2« (10~5 M) and PGE2 (10~7 M) increased the incorporation of [3H]thymidine into the acid-insoluble fraction of bone at 24 h (Table 2). The effects of PGF2a, but not those of PGE2, were blocked by both cortisol and indomethacin. The uptake of acid-soluble thymidine was not affected by PGE2 (data not shown). Because the effects of PGF2« were diminished by indomethacin and cortisol, we tested whether PGF2« might act by stimulating endogenous PG production in these cultures. Medium containing PGF2«, incubated without bone or with bones cultured in the presence of indomethacin, was used to correct for cross-reactivity of the antibody. In bones treated with 10~6 or 10"7 M PGF2a for 24 h, there was a significant increase in the concentration of iPGE2 (Table 3). In the presence of indomethacin, the values were the same as those in blank culture medium incubated without bones, which represented the crossreactivity of added PGF2a. To verify that PGF2a increased production of PGE2, bones were prelabeled with [3H]AA and treated with PGF2a at 10"5-10"7 M (Table 4). The chromatograms showed an increased amount of radioactivity in the PGE2 peak. The increase appeared to be maximal at 10~6 M and was not greater at 10~5 M. The release of [3H]AA was not increased. HPLC studies were also carried out with unlabeled cultures, and the amount of PGE2 was measured as the sum of the values by RIA of individual fractions in the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 01:48 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1990 Voll26«No2
EFFECTS OF PGF 2o ON BONE
1078
TABLE 1. Effects of PGF2a and PGE2 on bone weight, incorporation of [3H]proline into CDP and NCP, and PCS at 72 h in the presence or absence of cortisol (10"7 M) in cultured mouse parietal bones (dpm/Vg bone)
NCP (dpm/jig bone)
PCS
Bone wt
CDP
Control
283 ± 18
23 ± 1
19 ± 1
18.4 ± 0.5
PGF 2 a 10" 6 M 10" 6 M 10" 7 M
238 ± 18 243 ± 15 246 ± 18
22 ± 3 27 ± 2 23 ± 2
26 ± 2 27 ±2° 22 ± 2
13.1 ± 1.0° 15.6 ± 0.8 15.5 ± 0.9 •
Cortisol
240 ± 14 293 ± 18 270 ± 19 182 ± 166
11 ± 1° 18 ± 3 29 ± 2 10 ± 1"
25 ± 2 22 ± 3 29 ± 1° 14 ± 1
7.7 ± 0.3° 12.5 ± 0.6° 15.8 ± 0.5 11.5 ± 0.66
Cortisol + PGF2o, 10"5M 10"6M 10- 7 M
145 ± 7 159 ± 9 155 ± 14
18 ± 3
13 ±2 10 ± 1
21 ± 3 14 ± 1 14 ± 1
12.7 ± 0.9 14.2 ± 0.9 12.7 ± 1.0
Cortisol + PGE2 10"5M 10"6M 10"7M
330 ± 33° 306 ± 17° 235 ± 27
19 ±2 27 ±4° 28 ±2°
19 ± 2 26 ± 4 ° 26 ± 2°
15.3 ± 0.7 16.5 ± 0.5c 16.5 ± 0.7°
PGE2 10"6M 10"6M 10"7M
Values are the mean ± SE for 6-19 cultures. Significant effect of PG, P < 0.01. 6 Significant effect of cortisol, P < 0.01. c Significant effect of PG, P < 0.05. 0
TABLE 2. Effects of PGF2a and PGE2 on [3H]thymidine incorporation at 24 h in the presence or absence of cortisol (CORT; 10~7 M) or indomethacin (INDO; 10~6 M) in cultured mouse parietal bones Control Control
CORT
INDO
35 ± 4
12 ± 1
28 ± 3
100 ± 8° 78 ± 10° 66 ± 10
23 ± 3 19 ± 3 10 ± 1
26 ± 5 43 ± 3 ND
100 ± 8"
50 ±6°
66 ±8"
PGF 2a 10"5M 10"6M KT7M PGE 2 (10~7 M)
TABLE 3. Effect of PGF2a on iPGE2 concentration at 24 h in the presence or absence of indomethacin (INDO; 10~6 M) in cultured mouse parietal bones
Values are the mean ± SE for 10-16 cultures, expressed as disintegrations per ng bone. ND, Not determined. ° Significant effect of PG, P < 0.01. 6 Significant effect of PG, P < 0.05.
PGE2 peak. The mean PGE2 concentration for two samples of medium each was 1.5 nM for control, 2.3 nM at 10"7 M, and 4.7 nM at 10"6 M PGF 2a .
iPGE 2 cone. (nM)
Treatment Control PGF2a 10" 6 M 10" 7 M
With INDO
Blank
0.4 ± 0.1
ND
ND
8.4 ± 0.7° 4.2 ± 0.8fc
5.3 ± 0.1 0.3 ± 0.1
5.1 0.3
Values are the mean ± SE for 4-10 cultures. Blank values were obtained from medium incubated for 24 h without bones. ND, Not detectable (
Vol. 126, No. 2 Printed in U.S.A.
Effects of Prostaglandin F2« on Bone Formation and Resorption in Cultured Neonatal Mouse Calvariae: Role of Prostaglandin E2 Production* LAWRENCE G. RAISZt, CYNTHIA B. ALANDER, PAMELA M. FALL, AND HOLLIS A. SIMMONS Division of Endocrinology and Metabolism, University of Connecticut Health Center, Farmington, Connecticut 06032
labeling of CDP in the absence of cortisol and stimulated CDP labeling in the presence of cortisol. PGF2o increased thymidine incorporation into DNA, but the effect was smaller than that of PGE2 and was inhibited by indomethacin. These observations suggested that PGF2o might act in part by stimulating PGE2 production. By RIA, PGE2 concentrations were increased in the medium of bones treated with PGF2a, and this increase was blocked by indomethacin. By HPLC, bones prelabeled with [3H]arachidonic acid showed an increase in labeled PGE2 release, and RIA showed an increase in PGE2 after PGF2a treatment. These results indicate that PGF2a is a relatively weak agonist in bone compared to PGE2 and that some of the effects of PGF2a on bone resorption, formation, and cell replication may be mediated by an increase in endogenous PGE2 production. {Endocrinology 126: 1076-1079,1990)
ABSTRACT. Although most studies show that prostaglandin E2 (PGE2) is the most potent and effective of the prostanoids in bone, recent data in cell culture suggest that PGF2a may have unique effects, particularly on cell replication. The present study was undertaken to compare the effects of PGF 2 Q and PGE2 in cultured neonatal mouse parietal bones by simultaneous measurement of bone resorption as release of previously incorporated 46 Ca, bone formation as incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein, and DNA synthesis as incorporation of [3H]thymidine. PGF2a was less effective than PGE2 as a stimulator of bone resorption, and its effects were partially inhibited by indomethacin and markedly inhibited by glucocorticoids. In contrast, the resorptive response to PGE2 was unaffected by indomethacin and only partially inhibited by cortisol. PGF2a had little effect on bone formation, in contrast to the biphasic effect of PGE2, which inhibited
M
ANY studies in organ culture have indicated that prostaglandins of the E series (PGE) are potent stimulators of bone resorption and also have a biphasic effect on bone formation (1-3). In organ culture, PGF 2a was also found to stimulate resorption, but was less potent than PGE2. The effects of PGF2« on formation have not been extensively studied. Recently, studies in cell culture using either rat osteosarcoma cells (UMR 106) or cloned mouse cells with an osteoblastic phenotype (MC3T3-E1) have suggested that PGF 2a may be a potent mitogen in bone and that its effects could involve a separate receptor and second messenger system from that responsive to PGE 2 (4, 5).
by the incorporation of [3H]proline into collagenasedigestible (CDP) and noncollagen protein (NCP), and DNA synthesis by incorporation of [3H]thymidine in cultures of neonatal mouse parietal bones. In this system we found that PGF 2a was less potent than PGE2 and that its effects were diminished by treatment with indomethacin and cortisol. Based on these results, we examined the effect PGF 2a on PGE2 production and found an increase in medium PGE2 by RIA (iPGE2) and by analysis of [3H]PGE2 production in bones prelabeled with [3H] arachidonic acid ([3H]AA).
In the present study we have used an organ culture system in which bone resorption can be assessed by the release of previously incorporated 45Ca, bone formation
Timed mated pregnant mice (CD-I, Charles River, Wilmington, MA) were injected with 0.05 mCi 45Ca (New England Nuclear, Wilmington, DE) on the 16th day of gestation. Sevenday-old neonatal mice were killed, and the central portion of the parietal bone ( 2 x 3 mm) was dissected free of connective tissue and suture material and precultured for 18-24 h in BGJ medium (Gibco, Grand Island, NY) supplemented with 1 mg/ ml BSA (RIA grade, Sigma, St. Louis, MO) and containing 3 mM phosphate, 1 mM proline, and 100 Mg/ml freshly added ascorbic acid. The cultures were treated with PGE2, PGF2a,
Received September 8, 1989. * This work was supported by NIH Grants AR-18063 and AR-38933. A preliminary report of this work was presented at the combined meeting of the American Society for Bone and Mineral Research and the International Conferences on Calcium Regulating Hormones, Montreal, Quebec, Canada, September 1989. t To whom requests for reprints should be addressed.
Materials and Methods
1076
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EFFECTS OF cortisol (10 7 M), or indomethacin (10 6 M; Sigma). Cortisol and indomethacin were added during the preculture period. PGs and cortisol were dissolved in ethanol and added at a 0.1% final ethanol concentration, with a similar concentration in controls. PG treatment was carried out for 1-3 days, with daily medium changes. DNA synthesis was assessed by adding [3H] thymidine (5 /aCi/ml; New England Nuclear) for the last 2 h of a 24-h culture. Cold thymidine (10~5 M) was added to culture medium for these experiments. Acid-soluble [3H]thymidine was measured in trichloroacetic acid extracts of bone, and the residual bone was counted to assess DNA synthesis. In other cultures, [3H]proline (5 fiCi/m\; New England Nuclear) was added for the last 2 h of a 72-h culture, and its incorporation into CDP and NCP was measured as described previously (2). Percent collagen synthesis (PCS) was calculated after correction for the relative abundance of proline in CDP and NCP. 45 Ca was measured in the medium and trichloroacetic acid extracts of the bone to calculate percentage of 45Ca released. The medium iPGE2 concentration was measured by RIA (6). Two antibodies were used; the antibody from Dr. L. Levine of Brandeis University cross-reacted with PGF2« at 2%, while that kindly provided by Dr. N. H. L. Hunt (Canberra, Australia) cross-reacted only 0.4%. The latter was used for direct RIA of media to which PGF2«had been added. To assess PGE2 production further, two additional experiments were carried out. In one, bones were prelabeled with [3H]AA (10 j^Ci/ml) for 18 h. The bones were then washed for 2 h in fresh medium and cultured with PGF2o, for 24 h. The media were pooled from three cultures, acidified and extracted with ethyl acetate, and the products were separated on HPLC using a gradient system that separates PGF2« and PGE2by several fractions (7). HPLC profiles showed that the predominant labeled product was [3H] PGE2, as we have reported previously for rat calvariae (8). In the second experiment the media from unlabeled bones treated with PGF2« were pooled, extracted, and chromatographed on HPLC, and the PGE2 content of individual fractions was assessed by RIA. Statistical analysis was carried out by analysis of variance. Significance of differences was determined by post-hoc testing Using Bonferroni's method.
Results There was a significant increase in 45Ca release at 72 h with PGF 2a at 10"6 and 10"5 M, but not at lower concentrations (Fig. 1). In cultures treated with indomethacin the resorptive response was diminished and was significant only at 10~5 M. With cortisol the response to PGF2a was further diminished, but the small increase in 45Ca release at 10"5 M was significant (P < 0.05). PGE2 was more potent than PGF2« and produced a greater increase in 45Ca release. Stimulation of resorption was significant at 10~7 and 10~6 M (P < 0.01) and was similar in the presence or absence of indomethacin and decreased but still significant in the presence of cortisol. PGF 2a produced little change in the incorporation of proline into CDP or NCP. The only significant effects were an increase in NCP at 10~6 M and a decrease in
PGF2CT
ON BONE
Log PGF2-alpha [M]
1077
LogPGE2[M]
FIG. 1. Effect of PGF2aand PGE2on release of previously incorporated 45 Ca for neonatal mouse parietal bones at 72 h. Points are the means and vertical lines the SE for 6-36 cultures. Effects of PGF2a are significant (P < 0.01) at 10"5 and 10~6 M in the absence and at 10"6 M in the presence of indomethacin. Effects of PGE2 are significant (P < 0.01) at 10"6 and 10~7 M with or without cortisol or indomethacin.
PCS at 10"5 M (Table 1). In the presence of cortisol, which itself decreased labeling of CDP, PCS, and bone weight at 72 h, PGF2o had no significant effect. In contrast, PGE2 showed effects on bone formation in this model similar to those that we have reported previously in fetal rat calvaria (3). In the absence of cortisol, high concentrations inhibited incorporation of proline into CDP and decreased PCS. In the presence of cortisol, however, PGE2 stimulated incorporation of proline into CDP and NCP and increased bone weight. Both PGF2« (10~5 M) and PGE2 (10~7 M) increased the incorporation of [3H]thymidine into the acid-insoluble fraction of bone at 24 h (Table 2). The effects of PGF2a, but not those of PGE2, were blocked by both cortisol and indomethacin. The uptake of acid-soluble thymidine was not affected by PGE2 (data not shown). Because the effects of PGF2« were diminished by indomethacin and cortisol, we tested whether PGF2« might act by stimulating endogenous PG production in these cultures. Medium containing PGF2«, incubated without bone or with bones cultured in the presence of indomethacin, was used to correct for cross-reactivity of the antibody. In bones treated with 10~6 or 10"7 M PGF2a for 24 h, there was a significant increase in the concentration of iPGE2 (Table 3). In the presence of indomethacin, the values were the same as those in blank culture medium incubated without bones, which represented the crossreactivity of added PGF2a. To verify that PGF2a increased production of PGE2, bones were prelabeled with [3H]AA and treated with PGF2a at 10"5-10"7 M (Table 4). The chromatograms showed an increased amount of radioactivity in the PGE2 peak. The increase appeared to be maximal at 10~6 M and was not greater at 10~5 M. The release of [3H]AA was not increased. HPLC studies were also carried out with unlabeled cultures, and the amount of PGE2 was measured as the sum of the values by RIA of individual fractions in the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 01:48 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1990 Voll26«No2
EFFECTS OF PGF 2o ON BONE
1078
TABLE 1. Effects of PGF2a and PGE2 on bone weight, incorporation of [3H]proline into CDP and NCP, and PCS at 72 h in the presence or absence of cortisol (10"7 M) in cultured mouse parietal bones (dpm/Vg bone)
NCP (dpm/jig bone)
PCS
Bone wt
CDP
Control
283 ± 18
23 ± 1
19 ± 1
18.4 ± 0.5
PGF 2 a 10" 6 M 10" 6 M 10" 7 M
238 ± 18 243 ± 15 246 ± 18
22 ± 3 27 ± 2 23 ± 2
26 ± 2 27 ±2° 22 ± 2
13.1 ± 1.0° 15.6 ± 0.8 15.5 ± 0.9 •
Cortisol
240 ± 14 293 ± 18 270 ± 19 182 ± 166
11 ± 1° 18 ± 3 29 ± 2 10 ± 1"
25 ± 2 22 ± 3 29 ± 1° 14 ± 1
7.7 ± 0.3° 12.5 ± 0.6° 15.8 ± 0.5 11.5 ± 0.66
Cortisol + PGF2o, 10"5M 10"6M 10- 7 M
145 ± 7 159 ± 9 155 ± 14
18 ± 3
13 ±2 10 ± 1
21 ± 3 14 ± 1 14 ± 1
12.7 ± 0.9 14.2 ± 0.9 12.7 ± 1.0
Cortisol + PGE2 10"5M 10"6M 10"7M
330 ± 33° 306 ± 17° 235 ± 27
19 ±2 27 ±4° 28 ±2°
19 ± 2 26 ± 4 ° 26 ± 2°
15.3 ± 0.7 16.5 ± 0.5c 16.5 ± 0.7°
PGE2 10"6M 10"6M 10"7M
Values are the mean ± SE for 6-19 cultures. Significant effect of PG, P < 0.01. 6 Significant effect of cortisol, P < 0.01. c Significant effect of PG, P < 0.05. 0
TABLE 2. Effects of PGF2a and PGE2 on [3H]thymidine incorporation at 24 h in the presence or absence of cortisol (CORT; 10~7 M) or indomethacin (INDO; 10~6 M) in cultured mouse parietal bones Control Control
CORT
INDO
35 ± 4
12 ± 1
28 ± 3
100 ± 8° 78 ± 10° 66 ± 10
23 ± 3 19 ± 3 10 ± 1
26 ± 5 43 ± 3 ND
100 ± 8"
50 ±6°
66 ±8"
PGF 2a 10"5M 10"6M KT7M PGE 2 (10~7 M)
TABLE 3. Effect of PGF2a on iPGE2 concentration at 24 h in the presence or absence of indomethacin (INDO; 10~6 M) in cultured mouse parietal bones
Values are the mean ± SE for 10-16 cultures, expressed as disintegrations per ng bone. ND, Not determined. ° Significant effect of PG, P < 0.01. 6 Significant effect of PG, P < 0.05.
PGE2 peak. The mean PGE2 concentration for two samples of medium each was 1.5 nM for control, 2.3 nM at 10"7 M, and 4.7 nM at 10"6 M PGF 2a .
iPGE 2 cone. (nM)
Treatment Control PGF2a 10" 6 M 10" 7 M
With INDO
Blank
0.4 ± 0.1
ND
ND
8.4 ± 0.7° 4.2 ± 0.8fc
5.3 ± 0.1 0.3 ± 0.1
5.1 0.3
Values are the mean ± SE for 4-10 cultures. Blank values were obtained from medium incubated for 24 h without bones. ND, Not detectable (
