0021-972X/90/7003-0647S02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright© 1990 by The Endocrine Society
Vol. 70, No. 3 Printed in U.S.A.
Arachidonic Acid Release from Cultured Human Amnion Cells: The Effect of Dexamethasone* FRANK A. POTESTIO AND DAVID M. OLSONf Departments of Pediatrics and Physiology, Lawson Research Institute, St. Joseph's Health Centre of London, University of Western Ontario, London, Ontario, Canada N6A 4 V2
ABSTRACT. Glucocorticoids inhibit prostaglandin (PG) synthesis in several cell types, presumably by inhibiting arachidonic acid (AA) deacylation from phospholipids. We studied the effects of glucocorticoids on cultured term human amnion cell AA release. Confluent monolayer cultures of amnion cells were adapted to serum-free medium, and phospholipids were labeled for 18 h with [14C]AA. The calcium ionophore A23187 (0.2-5.0 jtmol/L) stimulated [14C]AA release (up to 2.2-fold) in a doseand time-dependent manner. The apparent sources of the liberated [14C]AA were phosphatidylcholine and phosphatidylethanolamine. Pretreatment for 24 h with the synthetic glucocorticoid dexamethasone (0.1-1000 nmol/L) significantly inhibited (P < 0.01) basal (unstimulated) [14C]AA release by 69% in subsequent 1-h experiments. The sole apparent source of free [14C]AA during this inhibitory state was phosphatidylethanolamine. Dexamethasone pretreatment slightly inhibited (13%; P < 0.05) calcium ionophore-stimulated [14C]AA release; however, it was still 3.8-fold greater than basal release, suggesting that
the glucocorticoid effect on stimulated AA release was not biologically relevant. Further characterization of the glucocorticoid effect revealed that preincubation of the cultures with dexamethasone for as little as 20 min inhibited basal [14C]AA release. Furthermore, studies involving actinomycin-D and cycloheximide demonstrated that inhibition of RNA and protein synthesis failed to block the glucocorticoid inhibition of basal AA liberation. The glucocorticoid receptor antagonist RU 38486, alone or in the presence of dexamethasone, also inhibited unstimulated [14C]AA release. Cortisol, dehydroisoandrosterone sulfate, 17/?estradiol, and progesterone all inhibited basal [14C]AA liberation. We conclude that glucocorticoids inhibit unstimulated AA release from cultured amnion cells, but do not prevent calcium ionophore from stimulating a large increase in AA release. Furthermore, glucocorticoid attenuation of unstimulated AA liberation is nonspecific and independent of RNA and protein synthesis and may be due to a general membrane effect of the steroid moiety. (J Clin Endocrinol Metab 70: 647, 1990)
P
the concentration of cortisol in amniotic fluid increases throughout gestation, with an abrupt rise before the onset of labor (8, 9). Furthermore, recent reports have shown that glucocorticoids stimulate PGE2 synthesis by cultured amnion cells (10, 11). Subsequent studies from our laboratory have demonstrated that this effect occurs via a receptor-mediated mechanism (12) and suggest that it stimulates the de nouo synthesis of the enzyme PG endoperoxide-H synthase (13). These data are novel, in that glucocorticoids are primarily noted for their antiinflammatory actions, which may be mediated by the proteins lipocortins (14, 15). Interestingly, glucocorticoids induce lipocortin-I formation in amnion (11), a protein that in many cell types inhibits the deacylation of AA from phospholipids and, hence, diminishes subsequent PG production due to an inadequate precursor supply (16-19). In this investigation we determined whether the synthetic glucocorticoid dexamethasone modulates the release of AA from human amnion cells at term.
rostaglandin E2 (PGE2) synthesis by human amnion increases with the onset of labor and is thought to participate in the initiation and maintenance of parturition (1-4). The mobilization of arachidonic acid (AA), the eicosanoid precursor, and its subsequent conversion to PGs are the key events in the enhanced production of PGE2 by amnion at term (1-4). Hence, an understanding of the mechanisms by which these two processes are regulated in amnion is essential to comprehend the regulation of parturition initiation in women. The glucocorticoid cortisol is one factor among many that may promote increased PG production at term in women. This concept is supported by several lines of evidence which suggest that the amnion is a glucocorticoid target organ at term. Firstly, amnion tissue contains high affinity glucocorticoid-binding sites with the characteristics of steroid hormone receptors (5-7). Secondly, Received August 3,1989. Address all correspondence and requests for reprints to: David M. Olson, Ph.D., Lawson Research Institute, St. Joseph's Health Centre of London, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. * This work was supported by the March of Dimes Birth Defects Foundation (Basic Research Grant 1-1122), the Medical Research Council of Canada (Grant MA-8557), and the Lawson Research Institute (studentship to F.A.P.). t Scholar of the Medical Research Council.
Materials and Methods Materials [1-14C]AA (SA, 58.3 mCi/mmol) in toluene was obtained from Amersham (Mississuaga, Ontario, Canada). The following 647
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POTESTIO AND OLSON
648
items were purchased from the Sigma Chemical Co. (St. Louis, MO): collagenase (type IV), soybean trypsin inhibitor (type Us), BSA (essentially fatty acid free, prepared from fraction V), HEPES, cycloheximide, actinomycin-D, dexamethasone, cortisol, dehydroisoandrosterone-3-sulfate, 17/?-estradiol, progesterone, and dimethylsulfoxide. Trypsin (1:250) was purchased from Gibco (Grand Island, NY). Powdered tissue culture medium (Dulbecco's Minimum Essential Medium, with glutamine, without sodium bicarbonate; Ham's F-12 modified, with glutamine, without sodium bicarbonate) and heat-inactivated fetal bovine serum were the products of Flow Laboratories, Inc. (McLean, VA). Calcium ionophore A23187 was supplied by Boehringer Mannheim (Dorval, Quebec, Canada). RU 38486 was acquired from Roussel-UCLAF (Romainville, France). Phospholipid and neutral lipid thin layer chromatography (TLC) standards were purchased from Serdary Research Laboratories (London, Ontario, Canada). TLC plates used were preadsorbent Si 250-PA(19C) from J. T. Baker Chemical Co. (Phillipsburg, NJ). All solvents were A.C.S. grade, and all other chemicals were reagent grade. Dispersal and culture of amnion cells The placentas (n = 14) used in this investigation were from uncomplicated pregnancies and were obtained at term after vaginal delivery after the spontaneous onset of labor. The reflected portion of the amnion was isolated and manually peeled away from the adjoining tissues, chorion, and decidua. After separation, the amnion was placed in Dulbecco's modified phosphate-buffered saline (20) containing 80 mg gentamicin sulfate/L. The tissue was washed in Dulbecco's modified phosphate-buffered saline and cut into pieces approximately 4 cm2. The tissue pieces were then incubated twice at 37 C in 12.5 mL collagenase solution (1200 U/mL buffer containing 4 mg BSA/ mL and 0.1 mg soybean trypsin inhibitor/mL) for 15 and 30 min, respectively. The buffer consisted of a 1:1 mixture of Ham's F-12 Modified Medium (Ham's F12) and Dulbecco's Minimum Essential Medium supplemented with 80 mg gentamicin sulfate/L and 15 mmol HEPES/L, pH 7.4. After the incubations in collagenase, the tissue pieces were washed, suspended, and centrifuged (600 X g for 10 min) twice in buffer. The amnion cells were liberated from the epithelial sheets by further digestion for 30 min at 37 C with 0.1 mg trypsin/mL dissolved in buffer. Trypsin was inactivated by dilution with a series of washes, suspensions, and centrifugations (200 X g for 10 min) sequentially in buffer supplemented with 0.1 mg trypsin inhibitor/mL and 4 mg BSA/mL, then buffer alone, and finally culture medium. Optimal dispersion was attained by aspirating and expelling the cell suspensions approximately six times through an 18-gauge spinal needle. Viability was determined using 0.05% trypan blue exclusion staining (21) and was consistently found to be greater than 95%. Amnion cells were seeded into plastic 24-well culture plates (Linbro, Flow Laboratories, Inc.) or 25-cm2 tissue culture flasks (Corning, NY) at a density of 1.0 X 105 cells/cm2. The cells were cultured in a 1:1 mixture of Ham's F12 and Dulbecco's Minimum Essential Medium supplemented with 10% heatinactivated fetal bovine serum and 20 mg gentamicin sulfate/ mL. The cultures were maintained in an atmosphere of 94%
JCE & M • 1990 Vol 70 • No 3
N2, 5% CO2, and 1% O2 at 37 C to attain an oxygen tension in the medium (35-36 mm Hg) that approached the pO2 of amniotic fluid (2-15 mm Hg) (22). The growth medium of the amnion cultures was changed every second day until confluence was reached between 6-10 days. Under phase contrast microscopy, the cells appeared as a homogeneous sheet of epithelial cells. Before experimentation, confluent monolayer cultures were conditioned to serum-free pseudoamniotic fluid (PsAF) containing 0.1 mg BSA/mL (23) in the following manner. Two days before an experiment, the growth medium over the cultures was replaced with medium containing 1% heat-inactivated fetal bovine serum. The following day, the cultures were washed with and then incubated in serum-free medium for 10 h. Radiolabeled AA ([14C]AA) (0.02 nCi/mL PsAF) was incorporated into phospholipids for 18 h. Unincorporated radioactivity was aspirated, and the cells were subsequently washed three times with PsAF to remove any [14C]AA that remained attached to the extracellular surface of the monolayer. Approximately 81 ± 5% of the radioactivity remained incorporated in the cells after this procedure (0.24 nmol [14C]AA/well). During the subsequent steroid treatment periods, control cultures were incubated in vehicle (
Vol. 70, No. 3 Printed in U.S.A.
Arachidonic Acid Release from Cultured Human Amnion Cells: The Effect of Dexamethasone* FRANK A. POTESTIO AND DAVID M. OLSONf Departments of Pediatrics and Physiology, Lawson Research Institute, St. Joseph's Health Centre of London, University of Western Ontario, London, Ontario, Canada N6A 4 V2
ABSTRACT. Glucocorticoids inhibit prostaglandin (PG) synthesis in several cell types, presumably by inhibiting arachidonic acid (AA) deacylation from phospholipids. We studied the effects of glucocorticoids on cultured term human amnion cell AA release. Confluent monolayer cultures of amnion cells were adapted to serum-free medium, and phospholipids were labeled for 18 h with [14C]AA. The calcium ionophore A23187 (0.2-5.0 jtmol/L) stimulated [14C]AA release (up to 2.2-fold) in a doseand time-dependent manner. The apparent sources of the liberated [14C]AA were phosphatidylcholine and phosphatidylethanolamine. Pretreatment for 24 h with the synthetic glucocorticoid dexamethasone (0.1-1000 nmol/L) significantly inhibited (P < 0.01) basal (unstimulated) [14C]AA release by 69% in subsequent 1-h experiments. The sole apparent source of free [14C]AA during this inhibitory state was phosphatidylethanolamine. Dexamethasone pretreatment slightly inhibited (13%; P < 0.05) calcium ionophore-stimulated [14C]AA release; however, it was still 3.8-fold greater than basal release, suggesting that
the glucocorticoid effect on stimulated AA release was not biologically relevant. Further characterization of the glucocorticoid effect revealed that preincubation of the cultures with dexamethasone for as little as 20 min inhibited basal [14C]AA release. Furthermore, studies involving actinomycin-D and cycloheximide demonstrated that inhibition of RNA and protein synthesis failed to block the glucocorticoid inhibition of basal AA liberation. The glucocorticoid receptor antagonist RU 38486, alone or in the presence of dexamethasone, also inhibited unstimulated [14C]AA release. Cortisol, dehydroisoandrosterone sulfate, 17/?estradiol, and progesterone all inhibited basal [14C]AA liberation. We conclude that glucocorticoids inhibit unstimulated AA release from cultured amnion cells, but do not prevent calcium ionophore from stimulating a large increase in AA release. Furthermore, glucocorticoid attenuation of unstimulated AA liberation is nonspecific and independent of RNA and protein synthesis and may be due to a general membrane effect of the steroid moiety. (J Clin Endocrinol Metab 70: 647, 1990)
P
the concentration of cortisol in amniotic fluid increases throughout gestation, with an abrupt rise before the onset of labor (8, 9). Furthermore, recent reports have shown that glucocorticoids stimulate PGE2 synthesis by cultured amnion cells (10, 11). Subsequent studies from our laboratory have demonstrated that this effect occurs via a receptor-mediated mechanism (12) and suggest that it stimulates the de nouo synthesis of the enzyme PG endoperoxide-H synthase (13). These data are novel, in that glucocorticoids are primarily noted for their antiinflammatory actions, which may be mediated by the proteins lipocortins (14, 15). Interestingly, glucocorticoids induce lipocortin-I formation in amnion (11), a protein that in many cell types inhibits the deacylation of AA from phospholipids and, hence, diminishes subsequent PG production due to an inadequate precursor supply (16-19). In this investigation we determined whether the synthetic glucocorticoid dexamethasone modulates the release of AA from human amnion cells at term.
rostaglandin E2 (PGE2) synthesis by human amnion increases with the onset of labor and is thought to participate in the initiation and maintenance of parturition (1-4). The mobilization of arachidonic acid (AA), the eicosanoid precursor, and its subsequent conversion to PGs are the key events in the enhanced production of PGE2 by amnion at term (1-4). Hence, an understanding of the mechanisms by which these two processes are regulated in amnion is essential to comprehend the regulation of parturition initiation in women. The glucocorticoid cortisol is one factor among many that may promote increased PG production at term in women. This concept is supported by several lines of evidence which suggest that the amnion is a glucocorticoid target organ at term. Firstly, amnion tissue contains high affinity glucocorticoid-binding sites with the characteristics of steroid hormone receptors (5-7). Secondly, Received August 3,1989. Address all correspondence and requests for reprints to: David M. Olson, Ph.D., Lawson Research Institute, St. Joseph's Health Centre of London, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. * This work was supported by the March of Dimes Birth Defects Foundation (Basic Research Grant 1-1122), the Medical Research Council of Canada (Grant MA-8557), and the Lawson Research Institute (studentship to F.A.P.). t Scholar of the Medical Research Council.
Materials and Methods Materials [1-14C]AA (SA, 58.3 mCi/mmol) in toluene was obtained from Amersham (Mississuaga, Ontario, Canada). The following 647
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 23:01 For personal use only. No other uses without permission. . All rights reserved.
POTESTIO AND OLSON
648
items were purchased from the Sigma Chemical Co. (St. Louis, MO): collagenase (type IV), soybean trypsin inhibitor (type Us), BSA (essentially fatty acid free, prepared from fraction V), HEPES, cycloheximide, actinomycin-D, dexamethasone, cortisol, dehydroisoandrosterone-3-sulfate, 17/?-estradiol, progesterone, and dimethylsulfoxide. Trypsin (1:250) was purchased from Gibco (Grand Island, NY). Powdered tissue culture medium (Dulbecco's Minimum Essential Medium, with glutamine, without sodium bicarbonate; Ham's F-12 modified, with glutamine, without sodium bicarbonate) and heat-inactivated fetal bovine serum were the products of Flow Laboratories, Inc. (McLean, VA). Calcium ionophore A23187 was supplied by Boehringer Mannheim (Dorval, Quebec, Canada). RU 38486 was acquired from Roussel-UCLAF (Romainville, France). Phospholipid and neutral lipid thin layer chromatography (TLC) standards were purchased from Serdary Research Laboratories (London, Ontario, Canada). TLC plates used were preadsorbent Si 250-PA(19C) from J. T. Baker Chemical Co. (Phillipsburg, NJ). All solvents were A.C.S. grade, and all other chemicals were reagent grade. Dispersal and culture of amnion cells The placentas (n = 14) used in this investigation were from uncomplicated pregnancies and were obtained at term after vaginal delivery after the spontaneous onset of labor. The reflected portion of the amnion was isolated and manually peeled away from the adjoining tissues, chorion, and decidua. After separation, the amnion was placed in Dulbecco's modified phosphate-buffered saline (20) containing 80 mg gentamicin sulfate/L. The tissue was washed in Dulbecco's modified phosphate-buffered saline and cut into pieces approximately 4 cm2. The tissue pieces were then incubated twice at 37 C in 12.5 mL collagenase solution (1200 U/mL buffer containing 4 mg BSA/ mL and 0.1 mg soybean trypsin inhibitor/mL) for 15 and 30 min, respectively. The buffer consisted of a 1:1 mixture of Ham's F-12 Modified Medium (Ham's F12) and Dulbecco's Minimum Essential Medium supplemented with 80 mg gentamicin sulfate/L and 15 mmol HEPES/L, pH 7.4. After the incubations in collagenase, the tissue pieces were washed, suspended, and centrifuged (600 X g for 10 min) twice in buffer. The amnion cells were liberated from the epithelial sheets by further digestion for 30 min at 37 C with 0.1 mg trypsin/mL dissolved in buffer. Trypsin was inactivated by dilution with a series of washes, suspensions, and centrifugations (200 X g for 10 min) sequentially in buffer supplemented with 0.1 mg trypsin inhibitor/mL and 4 mg BSA/mL, then buffer alone, and finally culture medium. Optimal dispersion was attained by aspirating and expelling the cell suspensions approximately six times through an 18-gauge spinal needle. Viability was determined using 0.05% trypan blue exclusion staining (21) and was consistently found to be greater than 95%. Amnion cells were seeded into plastic 24-well culture plates (Linbro, Flow Laboratories, Inc.) or 25-cm2 tissue culture flasks (Corning, NY) at a density of 1.0 X 105 cells/cm2. The cells were cultured in a 1:1 mixture of Ham's F12 and Dulbecco's Minimum Essential Medium supplemented with 10% heatinactivated fetal bovine serum and 20 mg gentamicin sulfate/ mL. The cultures were maintained in an atmosphere of 94%
JCE & M • 1990 Vol 70 • No 3
N2, 5% CO2, and 1% O2 at 37 C to attain an oxygen tension in the medium (35-36 mm Hg) that approached the pO2 of amniotic fluid (2-15 mm Hg) (22). The growth medium of the amnion cultures was changed every second day until confluence was reached between 6-10 days. Under phase contrast microscopy, the cells appeared as a homogeneous sheet of epithelial cells. Before experimentation, confluent monolayer cultures were conditioned to serum-free pseudoamniotic fluid (PsAF) containing 0.1 mg BSA/mL (23) in the following manner. Two days before an experiment, the growth medium over the cultures was replaced with medium containing 1% heat-inactivated fetal bovine serum. The following day, the cultures were washed with and then incubated in serum-free medium for 10 h. Radiolabeled AA ([14C]AA) (0.02 nCi/mL PsAF) was incorporated into phospholipids for 18 h. Unincorporated radioactivity was aspirated, and the cells were subsequently washed three times with PsAF to remove any [14C]AA that remained attached to the extracellular surface of the monolayer. Approximately 81 ± 5% of the radioactivity remained incorporated in the cells after this procedure (0.24 nmol [14C]AA/well). During the subsequent steroid treatment periods, control cultures were incubated in vehicle (
