Effect of parathyroid hormone on arachidonic acid metabolism in mouse osteoblasts: permissive action of dexamethasone.

001:~-~~2~/9~/130~-0s92$03.00/0 Endocrinology Copyright 8~ 1992 by The

Endocrine

Vol. Printed

Society

Effect of Parathyroid Hormone on Arachidonic Metabolism in Mouse Osteoblasts: Permissive Action of Dexamethasone FRANCOISE

SUAREZ

AND

CAROLINE

CNRS UA 583, HGpital Necker Enfants

Malades

130, No. 2 in U.S.A.

Acid

SILVE et Universite

Paris

V, Paris, France

resulted in a significant increase in free AA within the cells (mean + SE, +142 -t 11%; n = 9) and a short-lived change in the distribution of AA within cellular phospholipids (phosphatidylethanolamine, -63 + 3%; phosphatidylinositol, +168 f 7%; phosphatidylserine, +296 f 50%; sphingomyelin, +220 + 20%; lysophosphatidylcholine, +634 + 31%; mean f SE; n = 3), despite the fact that no changes in PTH-induced CAMP production were observed. Because the release of free AA is an essential step in the production of eicosanoid metabolites that could act as second messengers, these findings suggest that corticosteroid treatment may activate signal transduction pathways for PTH which are latent in untreated cells, and thereby explain at least in part the profound effects of corticosteroids on osteoblast function. (Endocrinology 130: 592-598, 1992)

ABSTRACT. We examined the regulation of arachidonic acid (AA) metabolism in primary cultures of mouse osteoblasts under steady state and after acute stimulation by PTH. Both dexamethasone-treated and untreated cells were evaluated, as glucocorticoids are known to modulate some actions of PTH in osteoblasts and to affect arachidonic acid metabolism in cells in general. Cells were labeled with [“H]AA for 3 h, followed by a 20-h equilibrium period, then exposed to 10mR M PTH for different time periods ranging from 2-60 min. The results showed that although osteoblasts maintained in vitro in the absence of dexamethasone were responsive to PTH, as measured by CAMP production (50-fold increase), PTH had no effect on the distribution of AA in phospholipids and did not induce the release of free AA from phospholipid pools. After 7-day treatment of the cells with 4 x lo-? M dexamethasone, 15-min PTH stimulation

A

RACHIDONIC acid (AA), as the precursor of the biologically active eicosanoid metabolites, is of key importance in cellular activation (1, 2). The level of free AA in cells is tightly controlled and results from a balance between the liberation of the AA from its esterified form in glycerophospholipids by phospholipases and its reesterification into phopspholipids by acyltransferases(3). The enzymatic pathways involved in these processesand the hormonal regulation of these pathways are still the subject of controversy and may depend on the cell type and/or the degree of cell differentiation or maturation. The involvement of AA metabolism in the regulation of bone and mineral metabolism is well established (4). Endogenous production of prostaglandins has been described in bone organ cultures and monolayer cultures of osteoblasts, the bone-forming cells. Differences in the amount and types of the prostanoid synthesized by these cells have been reported and have lead to the suggestion that osteoblast subgroups with specialized features exist (5, 6). Differences in the regulation of prostanoid syn-

thesis have also been reported in different models. Stimulation of de nouo synthesis and release of prostaglandins by PTH has been suggested in chick osteoblasts (6), but was not found in rat osteosarcoma cells (5). Although prostaglandin synthesis by osteoblast-like cells has been demonstrated, little information is available concerning AA metabolism in these cells or its regulation by calciotropic hormones. The present work was undertaken to gain information on the regulation of AA metabolism in primary cultures of osteoblasts under steady state and after acute stimulation by PTH. Both dexamethasone-treated and untreated cells were evaluated, as glucocorticoids are known to modulate some actions of PTH in osteoblasts (7) and to affect AA metabolism in cells in general (8).

Materials

and Methods

Osteoblast cultures

Osteoblastswere isolated from the calvaria of newborn mice (Charles River, Saint Aubin les Elbeuf, Fance), as previously described(9). Cells were plated at 2 x lo4 cells/cm’ in lo-cm’ tissue culture dishes containing 12 ml Dulbecco’s Modified Eagle’s Medium supplemented with 100 U/ml penicillin G, 50 pg/ml streptomycin, 1 mM glutamine (complete medium), and 20% fetal calf’ serum and cultured at 37 C in 95% air-5% CO,.

Received September 20, 1991. Address all correspondance and requests for reprints to: Caroline Silve, CNRS UA 583, Tour Technique 6Gme &age, HGpital Necker

Enfants Malades, 149 rue de Sivres, 75743 Paris Cedex, France. 592

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 02:26 For personal use only. No other uses without permission. . All rights reserved.

EFFECT

OF PTH

ON AA METABOLISM

The medium was replaced after 48 h and every third day thereafter with complete medium containing 10% fetal calf serum.

of cells with dexamethasone

Pretreatment

For some experiments, 5 ~1 lo-” M (4 X lo-’ M final concentration) dexamethasone (Sigma Chemical Co., St. Louis, MO) in ethanol were added to subconfluent cultures (5-6 days of culture), and cells were cultured for an additional 7 days. Fresh dexamethasone was added at each medium change. Control cultures, maintained in parallel, received 5 ~1 ethanol alone. Labeling

studies

To label the cells with [‘%H]AA or [“Hloleic acid ([“HIOA), the culture medium was removed, and the cultures were washed twice with 1 ml complete medium containing 1 mg/ml fatty acid-free BSA (Sigma; labeling medium) and incubated at 37 C in air-5% CO, in 750 ~1 labeling medium containing 0.5 &i/ ml [“H]AA or [“H]OA (Amersham, Arlington Heights, IL; SA, 200 and 5 Ci/mmol, respectively, for AA and OA)/culture dish. After 3 h, the medium was removed for determination of nonincorporated radioactivity, and culture dishes were washed twice with 1 ml labeling medium. Cultures were then incubated with 1 ml/dish complete medium containing 1 mg/ml BSA fraction V (Sigma) for 20 h at 37 C in air-5% CO,. Dexamethasone or ethanol was added, as described above, in the appropriate dishes. Stimulation of the cells by agonists was performed without additional medium change. Preliminary experiments demonstrated that this labeling protocol gives an optimum and stable incorporation of [“H]AA into cellular lipids. Stimulation

of osteoblasts by agonists

Radiolabeled cells, treated or not with dexamethasone, were stimulated at 37 C for varying times by adding 10 ~1 lo-” M human (h) PTH-(1-34) (Sigma) in 10 mM acetic acid-0.1% BSA (lo-” M final concentration), 5000 U phospholipase-As (PLA,; Sigma, no. 9279) dissolved in 10 ~1 H,O (50 mu/ml final concentration), or 10 ~1 10 mM acetic acid-0.1% BSA or HZO. At the end of the incubation, the medium was removed and centrifuged (2500 rpm; 15 min) to eliminate cellular debris, and aliquots of the medium (50 ~1) were used to determine total radioactivity. The remaining medium was frozen at -20 C for lipid extraction. To extract cellular phospholipids, 1 ml icecold methanol was added directly to the dishes. After 1 h at 4 C, the methanol was transferred to a 12 x 75-mm glass tube, each dish was washed again with 1 ml ice-cold methanol, and the two washes were combined. Lipid

analysis

Cellular lipids were extracted immediately to avoid uncontrolled catabolism. Extraction was performed according to the method of Bligh and Dyer (10). Briefly, 2 ml CHCl, and 1.5 ml HZ0 were added to the 2 ml methanol and mixed for 20 sec. After removal of the first CHC& extract, the remaining methanol-water phase was acidified with HCl (final concentration, 0.01 N) and extracted two additional times. The three CHCl,, extracts were combined, dried on a rotary evaporator, and

IN OSTEOBLASTS

593

stored at -20 C. [“HIAA- or [“H]OA-labeled phospholipids were separated by TLC. To do so, the dried samples were reconstituted in 200 ~1 chloroform-methanol (2:l); 20 ~1 of the reconstituted samples were spotted on 20 x 20-cm plastic Silicagel 60 plates (Merck, Darmstadt, Germany). The plates were run in one dimension using a methylacetate-l-propanol-chloroform-methanol-O.25% KC1 (25:25:25:10:9, by vol) solvent system at room temperature. The chromatogram was divided, and each lane was cut into 16 bands of 1 cm. The bands were mixed with scintillation fluid (Picofluor, Packard, Rungis, France), and radioactivity was determined by scintillation spectroscopy. Phospholipids were identified by comparing their migration to that of standards. This system allows the complete separation of phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM), and lysophosphatidylcholine (LPC) (11). For determination of free [“HIAA, the plates were developed in one dimension with the solvent system obtained from the upper phase of a mixture of ethylacetate-isooctane-acetic acidHZ0 (110:50:20:100, by vol). This system allows the complete separation of AA and its eicosanoid derivatives (12). CAMP cellular

content

Cells were cultured and treated with dexamethasone as described above, except that cells were grown in 24-well culture dishes. CAMP production was determined as previously described (13). Briefly, culture medium was replaced with 250 pl/ well Dulbecco’s Modified Eagle’s Medium containing 0.1% of BSA, 1 mM isobutylmethylxanthine (Sigma), and 5 ~1 of a solution containing 0.75-250 ng hPTH-(1-34) dissolved in 10 mM acetic acid-0.1% BSA (final concentration of PTH, 0.8240 nm) or solvent alone. After a lo-min incubation at room temperature, the cells were washed three times with 1 ml icecold PBS, and cellular CAMP was extracted twice using 1 ml ethanol. CAMP was measured using a protein binding assay (14). Total protein present in the cell layer of the cultures was determined by the method of Lowry et al. (15). Results are expressed as picomoles of CAMP per 100 pg protein. Statistical

analysis

Unless otherwise stated, results are the mean + SE of three to nine experiments, in which three to five determinations per point were obtained. The data on the effect of dexemathasone on the distribution of [“H]AA in cellular phospholipids were analyzed by two-way analysis of variance, and results were compared by modified t test (16). The data on the effect of PTH on the distribution of [“H]AA in cellular phospholipids as a function of time were analyzed by one-way analysis of variance, and individual comparaisons between pairs were made by Dunnett’s method. When only two groups were compared, the data were analyzed by Student’s unpaired two-tailed t test.

Results

fH]AA incorporation and distribution osteoblasts: effect of dexamethasone

in mouse

After a 3-h incubation h equilibrium period,

followed by a 20of the initially

with [“HIAA, the proportions


EFFECT

594

OF PTH

ON AA METABOLISM

added radioactivity that was incorporated in cells were 70 + 2% and 67 f 2%, respectively, for untreated and dexamethasone-treated mouse osteoblast cultures (n = 3). Essentially all of the radioactivity extracted as lipids from the cell layer cultures not receiving dexamethasone was recovered as phospholipids (94 f 2%, corresponding to 21 f 1 X lo4 cpm). The distribution of radioactivity in cellular phospholipids, in decreasing order, was as follows: PE, PC, PI, PS, PA, LPC, and SM (Table 1). Dexamethasone pretreatment did not change the total incorporation of [3H]AA into cellular phospholipids (96 +- l%, corresponding to 20 f 3 x lo4 cpm; P > 0.2 compared to cultures not receiving dexamethasone), but significantly increased the incorporation of [3H]AA into PC (+140 f 12%; n = 3; P < 0.01) and decreased the incorporation into PI (-35 f 6%; n = 3; P < 0.05) compared to those in untreated cultures (Table 1). Incorporation into the other phospholipids was not significantly changed by dexamethasone treatment. Effect of PTH on pH]AA distribution between the phospholipids in mouse osteoblasts treated or not with dexamethasone

In the absence of dexamethasone treatment, the distribution of [3H]AA between the various cellular phospholipids was not significantly changed after exposure to lo-’ M PTH (Fig. 1). In cells pretreated with dexamethasone, however, 15 min after PTH stimulation, a dramatic decrease in the amount of [3H]AA recovered with PE was observed, and this was associated with a reciprocal increase in the amount of [3H]AA recovered as PI, PS, SM, and LPC (Fig. 2, A and B). Only the recovery of [3H]AA into PA (not shown) and PC (Fig. 2B) was not significantly different in dexatreated cells before and after PTH stimulation. The time course for the effect of PTH on the distribution of [3H]AA between the cellular phospholipids confirmed the absence of a PTH effect in dexamethasone-untreated cells, even at time points earlier or later than 15 min. In dexamethasone-treated cells, PTH-induced changes in the distribution of phospholipids reached a maximum at 15 min, but had returned to baseline levels after 30 min (Fig. 2, A and B). TABLE

1. Effect

of dexamethasone

Control Dexamethasone

on the distribution

of [3H]AA

between

IN OSTEOBLASTS

Distribution of fH]OA

Endo. Vol130.No

2

in cellular phospholipids

To evaluate the specificity of the effect of PTH on AA metabolism, we studied the effect of PTH on the distribution of OA, a fatty acid which, like AA, is largely incorporated at the sn-2-position of phospholipids. In contrast to the results obtained with AA, PTH stimulation (lo-’ M; 15 min) of nondexamethasone-treated or dexamethasone-treated cells did not change the distribution of [“H]OA in any phospholipids (Fig. 3). PH]AA

release in incubation medium and cells

Unstimulated cells. Under steady state conditions, 9,540 f 718 cpm (mean + SE; n = 9) were recovered in the medium of nondexamethasone-treated cells after 15-min incubation at 37 C in the presence of PTH diluent. When this radioactivity was separated by TLC using the solvent system that resolves AA and its eicosanoid metabolites, a single peak of free [3H]AA was detected, representing most of the total radioactivity that migrated from the origin. No other peak of radioactivity was observed, suggesting that under these conditions, [3H]AA was not present as eicosanoid derivatives of AA. Free [3H]AA represented 3.1 + 0.3% of the total radioactivity present in the cells. Dexamethasone treatment did not significantly change the amount of free [3H]AA present in the medium (11,160 + 1,306 cpm recovered in the medium) or cells (3.1 + 0.2% of total cellular radioactivity) compared to that observed in nondexamethasone-treated cells (P > 0.2 for both comparisons). Effects of PTH and exogenous PLA2. PTH

(lo-’ M; 15 min) did not significantly change the amount of total radioactivity released into the incubation medium from either untreated or dexamethasone-treated cells (Fig. 4). Furthermore, when the calcium concentration of the incubation medium was varied over the range of 0.5-1.5 mM, PTH-induced changes in the release of total radioactivity into the medium were not observed (data not shown). The effect of exogenous PLA, on the release of radioactivity and [3H]AA was also evaluated. Exogenous PLAz (50 mu/ml) did not change the release of radioactivity into the medium of cells not pretreated with dexamethasone, but significantly increased the amount

the various

phospholipids

from

mouse

osteoblasts

PE

PA

PI

PS

PC

SM

LPC

30.0 * 4.0 31.2 + 9.2

1.6 + 0.5 1.8 + 1.4

25.0 + 6.4 17.7 f 2.3”

7.2 + 1.8 5.6 + 1.5

27.4 + 4.8 37.3 +- 6.1*

0.8 + 0.1 0.9 f 0.9

1.3 + 0.8 1.4 + 0.6

Mouse osteoblasts were grown to subconfluence and 4 x 10e7 M dexamethasone or vehicle was added to the cultures. After 7 additional days in culture, cells were labeled with [SH]AA, the phospholipids were extracted, separated by TLC, and analyzed for radioactivity, as described in Materials and Methods. The results are expressed as a percentage of the total cellular radioactivity extracted in phospholipids and are the mean + SE of three experiments, with three to nine determinations per point. ’ P < 0.05 compared to control cells. * P < 0.01 compared to control cells.


EFFECT

PE

PA

PI

PS

OF PTH

PC

SM

ON AA METABOLISM

LPC

FIG. 1. Effect of PTH on the distribution of [3H]AA in phospholipids from mouse osteoblasts. Osteoblasts were labeled with [“H]AA and stimulated by hPTH-(1-34) (10-s M) for 15 min at 37 C. At the end of the stimulation period, phospholipids were extracted, separated by TLC, and analyzed for radioactivity, as described in Materials and Methods. Results are expressed as a percentage of the total cellular radioactivity extracted in phospholipids (PL) and represent the mean f SD of one experiment performed in triplicate. 0, Control cells; q , PTH-stimulated cells.

of radioactivity released in the medium from dexamethasone-treated cells (Fig. 4). TLC analysis of the medium demonstrated an increase in free [“H]AA (Fig. 5). Although PTH did not change the amount of [3H]AA released into the culture medium, addition of lo-” M PTH to dexamethasone-treated cells resulted in a significant increase in the amount of free [3H]AA present in the cells 15 min after stimulation (142 + 32%; corresponding to the time when the greatest modifications in the distribution of [3H]AA in cellular phospholipids were observed, Fig. 5). As was observed for PTH, the ionophore A23187 (lo-” M) was capable of inducing changes in the distribution of AA only in dexamethasone-treated cells (data not shown). Effect of dexamethasone on the stimulation of CAMP production by PTH

Dexamethasone treatment in concentrations ranging from 10-‘“-10-5 M did not significantly change the concentration of PTH required for half-maximal stimulation of CAMP production (-8 x lo-’ M) or the maximal CAMP production in response to PTH (300 f 15 and 280 + 10 pmol cAMP/lOO gg protein, respectively in nondexamethasone-treated cells and cells treated with 4 x lop7 M dexamethasone, respectively; Fig. 6). Dexamethasone treatment did not significantly change the protein concentrations of the cultures (370 f 52 and 327 + 14 pug protein/dish, respectively, in nondexamethasone-treated cells and cells treated with 4 x lop7 M dexamethasone, respectively; mean f SD; n = 18). Discussion

This study demonstrates that although osteoblasts maintained in vitro in the absence of dexamethasone are

IN OSTEOBLASTS

595

responsive to PTH, as measured by CAMP production, PTH has no effect on the distribution of AA in phospholipids and does not induce the release of free AA from phospholipid pools. After long term treatment of the cells with dexamethasone, PTH stimulation results in a significant increase in free AA within the cells and a short-lived change in the distribution of AA within cellular phospholipids, despite the fact that no changes in PTH-induced CAMP production are observed. Because the release of free AA is an essential step in the production of eicosanoid metabolites acting as second messengers, these findings suggest that corticosteroid treatment may activate signal transduction pathways for PTH that are latent in untreated cells and thereby explain at least in part the profound effects of corticosteroids on osteoblast function. In agreement with results obtained in other mammalian systems, we found that the bulk of AA in mouse osteoblasts was incorporated in glycerophospholipids, whereas only a low level of free cellular AA was present. Under steady state culture conditions, the metabolism of AA in these cells could not be altered by PTH, as illustrated by the absence of change in either the distribution of [“H]AA in cellular phospholipids or the release of AA in the cells or incubation medium. In contrast to the absence of PTH effect in nondexamethasone-treated cells, acute stimulation by PTH of dexamethasone-pretreated cells resulted in an increase in intracellular free AA and produced a striking redistribution of AA in cellular phospholipids (e.g. a decrease in PE-associated AA and a reciprocal increase in AA associated with PI, PS, SM, and LPC). These changes were reversible with time and appeared to be independent of the stimulation of CAMP production by PTH. In rat and human osteoblast systems, dexamethasone treatment can alter PTH stimulation of CAMP production (7, 13, 17). Thus, the lack of effect that we observed could be a characteristic of the mouse system. The changes in the distribution of AA among the different phospholipids were linked to each other, as the peak responses were observed at 15 min in all cases. No effect of PTH was observed when cells were labeled with [3H]OA, emphasizing the specificity of these changes for AA. PTH and AA metabolism

Two general pathways could be involved in the effects of PTH. Modifications of specific enzymes involved in phospholipid metabolism that are independent of AA metabolism (such as PE serine transferase) are unlikely to explain these results (18, 19). First, the decrease in [“H]AA incorporation into PE was associated with an increase in cellular free AA, suggesting that AA release was required for redistribuiton. Second, the changes in AA distribution were not observed for OA, a finding that


596

EFFECT

OF PTH

ON AA METABOLISM

Endo. Vol130.NoZ

IN OSTEOBLASTS

% -ii 15or =1

FIG. 2. Effect of PTH on the distribution of [3H]AA in phospholipids from mouse osteoblasts as a function of time. Osteoblasts, treated or not with dexamethasone (4 X 10m7 M; 7 days), were labeled with [3H]AA and stimulated by hPTH-(l-34) (lo-’ M) at 37 C for the indicated time. Phospholipids were extracted, separated by TLC, and analyzed for radioactivity, as described in Muteriuk and Methods. Results are expressed as a percentage of the values obtained in the absence of PTH in untreated (IY) and dexamethasone-treated (M) cultures. The dark and clear shaded areas indicate the SEM for samples from untreated and dexamethasone-treated cells, respectively. Results are the mean f SE of three experiments. *, P < 0.05; t*, P < 0.01; tt*, P < 0.001 (compared to cells not stimulated by PTH). PL, Phospholipids.

!i ‘d 2

200

PC

r

*_*

PI

150

8

i% 00

2 i

(z

100 5.

I

OIL A

15

30 Time

strongly supports the idea that the effects of PTH involved the action of an AA phospholipase. Indeed, our results are compatible with the activation of a phospholipase releasing AA, followed by the stimulation of acyl transferases resulting in the reincorporation of AA in phospholipids (3). The biochemical pathways involved in the release of AA in bone cells are poorly understood and could involve the activation of a PLC, followed by the action of a diacylglycerol lipase, the activation of a PLA1, followed by the action of a lysophospholipase or the activation of a PLAz (3). Because AA is almost exclusively incorporated in the sn-2 position in glycerophospholipids, PLAz is an attractive candidate to explain AA release. Corticosteroids and AA metabolism

Two general mechanisms could be implicated in the permissive action of dexamethasone on the mobilization

45 (min)

60

0

15

B

30 Time

45

60

bin)

of AA by PTH. First, differences in the ability of different phospholipid pools to release AA in response to hormonal stimulation have been described in other systems (20). Thus, it is possible that dexamethasone pretreatment promoted the incorporation of AA into phospholipid pools from which it is more easily mobilized. The fact that dexamethasone treatment had a significant effect on the steady state distribution of AA in cellular phospholipids is consistent with this idea, as is the observation that exogenous PLAz could only release AA from dexamethasone-treated cells. Alternatively, dexamethasone could influence the activity or regulation of enzymes required to liberate AA, such as PLA2, discussed above. The effects of dexamethasone on G-proteins, implicated in the coupling of receptors to effecters, could mediate these actions (17,22,23). In these regard, ,&subunits of G-proteins have recently been shown to directly activate PLAz (24), and cortico-


EFFECT

OF PTH ON AA METABOLISM

DEX

IN OSTEOBLASTS

-

PTH

DFX

m

m

PE

PA

PI

PS

PC

SM

597

+

PLA2

FIG. 4. Effects of PTH and PLA, on the release of radioactivity in the medium of cells treated or not with dexamethasone. Confluent osteoblasts were treated with dexamethasone (4 X 1O-7 M) or solvent for 7 days and labeled with [“HIAA, as described in Materials and Methods. After 15-min stimulation by PTH (10-s M) or PLA, (50 mu/ml) at 37 C, an aliquot of the incubation medium was counted to determine the radioactivity released in the medium. Results are expressed as a percentage of the amount (counts per min) released in the absence of PTH or PLA, in untreated (0) and dexamethasone-treated (M) cells and are the mean + SE of nine experiments. **t, P < 0.001 compared to unstimulated cells.

LPC

FIG. 3. Effects of dexamethasone pretreatment and PTH stimulation on the distribution of [3H]OA in phospholipids from mouse osteoblasts. Osteoblasts, treated or not with dexamethasone (DEX; 4 x 10m7 M; 7 days), were labeled with [“H]OA and stimulated by hPTH-(1-34) (lo-@ M) at 37 C for 15 min. Phospholipids were extracted, separated by TLC, and analyzed for radioactivity, as described in Materials and Methods. 0, Control; q , PTH-stimulated cultures. Results are expressed as a percentage of the total radioactivity in cellular phospholipids (PL) and are the mean + SE from one experiment, performed in triplicate.

steroid-induced changes in G-protein have been described in several systems (21-23), including bone cells (17). For example, a dexamethasone-induced increase in mRNA coding for P-subunits has been reported in adipocytes (23). Thus, it is conceivable that dexamethasone increases the synthesis of a G-protein or G-protein subunit in our system, which activates a PLA*, leading to coupling between the PTH receptor and AA release. It should be noted that the inhibition of PLAz activity by dexamethasone has been reported (8). Such inhibition is not universal. For example, in rat proximal-small-intestinal brush-border membranes, no significant alteration of PLAz activity was found after dexamethasone administration (25), and inhibition of prostaglandin Ip synthesis by dexamethasone without alteration of AA liberation has been reported in endothelial cells (26). In conclusion, PTH is able to stimulate AA metabolism in osteoblast-like cells. The expression of this transduc-

CELLS

MEDIUM

FIG. 5. Effects of PTH and exogenous PLA, on the amount of free (“H]AA present in incubation media and cells of cultures treated with dexamethasone. Confluent osteoblasts were treated with dexamethasone for 7 days, labeled with [3H]AA, and stimulated for 15 min at 37 C by lo-” M hPTH-(l-34) (0) or 50 mu/ml PLAZ (B). Free [“H]AA in the media and cells was extracted and analyzed, as described in Muterials and Methods. Results are the mean k SE of nine and six experiments, respectively, for PTH and PLA,. *, P < 0.05; ***, P < 0.001 (compared to nonstimulated cells).

tion pathway is independent of CAMP production and requires a permissive action of dexamethasone. Activation of this transduction pathway for PTH may be an important event mediating the actions of glucocorticoids on bone metabolism. Such interactions between long term treatment by steroid hormones and acute effects of peptide hormone on phospholipid metabolism might be of relevance in a variety of physiological and pathological processes.


598

EFFECT OF PTH ON AA METABOLISM

8. 9. 10. 11. 12.

~PTH

(l-34)

13. rig/ml

FIG. 6. Stimulation of CAMP production by increasing PTH concentrations in mouse osteoblasts treated or not with dexamethasone. Mouse osteoblasts were grown to subconfluence, and 4 X 10e7 M dexamethasone or vehicle was added to the cultures. After 7 additional days in culture, hPTH-(1-34) was added at the indicated concentrations, and after a lo-min incubation, cellular CAMP was extracted and assayed, as described in Materials and Methods. Results are the mean f SD of one representative experiment, performed in triplicate. q , Untreated cells; n , dexamethasone-treated cells.

Acknowledgments We wish to thank B. Rothut for helpful discussions, L. Touqui for his advice and careful reading of the manuscript, and R. Delaveyne for her help in statistical analysis.

14.

15. 16. 17. 18. 19. 20.

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Effect of metabolic acidosis on renal action of parathyroid hormone.

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Distal site of action of parathyroid hormone on phosphate reabsorption.

Effect of parathyroid hormone on urinary acidification.

Examination of the requirement for metabolism of parathyroid hormone in skeletal tissue before biological action.

Effect of tert-butyl hydroperoxide on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in rabbit platelets.

Effect of in vivo coal dust exposure on arachidonic acid metabolism in the rat alveolar macrophage.

Parathyroid Hormone Reprograms Osteoblast Metabolism.

Effect of indomethacin on arachidonic acid metabolism in human leukocytes stimulated ex vivo.

Parathyroid hormone, but not prostaglandin E2, changes the shape of osteoblasts maintained on bone in vitro.

The peripheral metabolism of parathyroid hormone.

Effect of experimental human magnesium depletion on parathyroid hormone secretion and 1,25-dihydroxyvitamin D metabolism.

Cytochrome P-450 metabolism of arachidonic acid.

The multiple pathways of arachidonic acid metabolism.

The effect of bovine parathyroid hormone withdrawal on MC3T3-E1 cell proliferation and phosphorus metabolism.

Arachidonic acid metabolism in airway epithelial cells.

The effect of growth hormone on parathyroid function in rats.

Arachidonic acid release from cultured human amnion cells: the effect of dexamethasone.

Effect of dexamethasone on parathyroid hormone (PTH) and PTH-related protein regulated phosphate uptake in opossum kidney cells.

Effect of parathyroid hormone on plasma prolactin in man.

Diethyldithiocarbamate (ditiocarb sodium) effect on arachidonic acid metabolism in human mononuclear cells. Glutathione peroxidase-like activity.

Effect of cortexolone on the feedback action of dexamethasone.

In vitro goldfish growth hormone responses to gonadotropin-releasing hormone: possible roles of extracellular calcium and arachidonic acid metabolism?

Vasodilatory action of arachidonic acid in humans following indomethacin treatment.