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Polyunsaturated Fatty Acids and

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Polyunsaturated Fatty Acids and Powered By Docstoc
					BIOLOGY OF REPRODUCTION 69, 780–787 (2003)
Published online before print 30 April 2003.
DOI 10.1095/biolreprod.102.015057


Polyunsaturated Fatty Acids and Bovine Interferon- Modify Phorbol Ester-Induced
Secretion of Prostaglandin F2 and Expression of Prostaglandin Endoperoxide
Synthase-2 and Phospholipase-A2 in Bovine Endometrial Cells1

Ricardo Mattos, Aydin Guzeloglu, Lokenga Badinga, Charles R. Staples, and William W. Thatcher2
Department of Animal Sciences, University of Florida, Gainesville, Florida 32611-0910

ABSTRACT                                                                        the pregnancy rates of beef cows at 30 days after artificial
   Embryonic mortality in cattle may occur because of inade-
                                                                                insemination from the 80% observed in the untreated con-
quate inhibition of uterine secretion of prostaglandin (PG) F2                  trols to 30%. Treating cows concomitantly with flunixin
mediated by bovine interferon- (bIFN- ). The objectives of the                  meglumine, an inhibitor of prostanoid synthesis, restored
present study were to determine whether polyunsaturated fatty                   pregnancy rates to 80% [2]. Another study [3] demonstrated
acids inhibit secretion of PGF2 from bovine endometrial cells                   that chronic treatment with PGF2 reduced the pregnancy
induced by stimulating protein kinase C with phorbol 12,13 di-                  rate when given between Days 5 and 8 of pregnancy and
butyrate (PDBu) and to investigate possible mechanisms of ac-                   not on Days 10–13 and 15–18. These results demonstrate
tion. Confluent cells were exposed for 24 h to 100 M of lin-                     that increased PG secretion during early pregnancy causes
oleic, arachidonic (AA; C20:4, n-6), linolenic (LNA; C18:3, n-3),               embryonic loss. In addition, the results support the hypoth-
eicosapentaenoic (EPA; C20:5, n-3), or docosahexaenoic (DHA;                    esis that reducing PG synthesis during early pregnancy re-
C22:6, n-3) acid. After incubation, cells were washed and stim-                 duces embryonic loss and improves pregnancy rates.
ulated with PDBu. The EPA, DHA, and LNA attenuated secretion                       Secretion of prostanoids, such as PGF2 , requires a cas-
of PGF2 in response to PDBu. The EPA and DHA were more                          cade of intracellular events, including activation of the
potent inhibitors than LNA. The EPA inhibited secretion of PGF2                 phospholipase (PL) C enzyme and activation of a calcium-
at 6.25 M. Secretion of PGF2 in response to PDBu decreased
                                                                                dependent PLA2 through a multiple antigenic peptide ki-
with increasing incubation time with EPA. Both bIFN- and EPA
inhibited secretion of PGF2 , and their inhibitory effects were
                                                                                nase-dependent pathway [4]. The activated PLA2 translo-
additive. The bIFN- , but not EPA, reduced the abundance of PG                  cates to the plasma membrane and cleaves fatty acids from
endoperoxide synthase-2 (PGHS-2) mRNA. Incubation with 100                      the sn-2 position of membrane phospholipids, to which a
  M EPA, DHA, or AA for 24 h followed by treatment with PDBu                    high proportion of polyunsaturated fatty acids (PUFAs),
did not affect concentrations of PGHS-2 and phospholipase A2                    such as arachidonic acid (AA), is esterified. Released AA
proteins. The EPA and DHA inhibit secretion of PGF2 through                     is converted to PGH2 by the PG endoperoxide H synthase
a mechanism different from that of bIFN- . The effect of EPA on                 (PGHS) enzymes. Two PGHS enzymes, PGHS-1 and
PGF2 secretion may be caused by competition with AA for                         PGHS-2, with similar structures and functions but different
PGHS-2 activity or reduction of PGHS-2 activity. The use of EPA                 expression patterns, have been described [5]. Prostaglandin
and DHA to inhibit uterine secretion of PGF2 and to improve                     H2 is subsequently reduced to PGF2 by PGF2 synthase or
embryonic survival in cattle warrants further investigation.                    is isomerized to other prostanoids of the 2 series, such as
conceptus, cytokines, female reproductive tract, implantation,                  PGE 2 [6].
uterus                                                                             The PLA2 and PGHS enzymes also are capable of pro-
                                                                                cessing other fatty acids, such as eicosapentaenoic acid
INTRODUCTION                                                                    (EPA), which is the precursor for synthesis of prostanoids
   A high proportion of embryonic loss occurs in cattle                         of the 3 series. Prostanoids of the 3 series, which include
during the first 3 wk of pregnancy [1]. This high proportion                     PGF3 and PGE3, have three double bonds in the carbon
of losses coincides with the period of embryonic inhibition                     chain, as opposed to the two double bonds found in pros-
of uterine prostaglandin (PG) F2 secretion and suggests                         tanoids of the 2 series. Increased availability of n-3 PUFAs,
that some loss may be occurring because certain embryos                         such as EPA and docosahexaenoic acid (DHA), in mem-
are unable to inhibit secretion of PGF2 . Therefore, strate-                    brane phospholipids could inhibit PGF2 secretion through
gies to further inhibit secretion of PGF2 may increase em-                      three mechanisms. First, EPA could displace AA, leading
bryonic survival. In a recent report, increased PG synthesis                    to increased synthesis of prostanoids of the 3 series at the
induced by oxytocin during Days 5–8 of pregnancy reduced                        expense of prostanoids of the 2 series, such as PGF2 . Sec-
                                                                                ond, EPA and another n-3 PUFA, DHA, may reduce ex-
1
 This research was supported in part by the NRI Competitive Grant Pro-          pression of the PGHS enzymes [7], which could make these
gram/USDA, grant 98-35203-6367. This is Florida Agricultural Experi-            enzymes less available and further reduce prostanoid syn-
mental Station Journal Series R-09552.                                          thesis. Third, the conversion of EPA into prostanoids of the
2
 Correspondence: William W. Thatcher, Department of Animal Sciences,            3 series is less efficient than the conversion of AA into
University of Florida. P.O. Box 11910, Gainesville, Florida 32611-0910.         prostanoids of the 2 series [8]. A lower efficiency of catal-
FAX: 352 392 5595; e-mail: thatcher@animal.ufl.edu
                                                                                ysis may result in reduced total prostanoid synthesis be-
                                                                                cause of insufficient converting capacity.
Received: 30 December 2002.                                                        Prostanoids of the 3 series are less bioactive [9], and
First decision: 19 January 2003.
Accepted: 22 April 2003.                                                        their role in ruminant luteolysis is currently unknown. Pros-
   2003 by the Society for the Study of Reproduction, Inc.                      taglandin F3 has only 25% affinity for the ovine luteal
ISSN: 0006-3363. http://www.biolreprod.org                                      PGF2 receptor when compared to PGF2 [10]. In addition,
                                                                          780
                                       FATTY ACIDS REGULATE PROSTAGLANDIN SECRETION                                                                 781

DHA is not a substrate for the PGHS enzymes but is a                         midified atmosphere containing 95% air and 5% CO2. On reaching con-
strong competitive inhibitor of their activity [11].                         fluence, cells were washed twice with HBSS at 37 C and cultured for an
                                                                             additional 24 h in culture medium devoid of serum with or without fatty
    The bovine interferon- (bIFN- ) attenuates secretion of                  acid. The 24-h incubation period with fatty acids was chosen based on
endometrial PGF2 both in vivo [12] and in vitro [13–16].                     reported effects of AA on PGF2 secretion from bovine endometrial ex-
Experimental approaches used to test the effects of bIFN-                    plants after incubation for 24 h [20]. At completion of this 24-h incubation
have included explants [13], primary cultures of endome-                     period (designated as 0 h), cells were washed again with HBSS to remove
trial cells collected on Days 1–4 [15] or on Day 15 of the                   fatty acids and exposed to serum-free medium mixed with appropriate
estrous cycle [14], and bovine immortalized endometrial                      treatments. In experiments 1–6, samples (250 l) were collected imme-
                                                                             diately before cell wash (0 h) and at 3 and 6 h after addition of PDBu.
(BEND) cells [16]. The BEND cells are a line of sponta-                      At 3 h, the volume removed was replaced by adding 250 l of medium
neously replicating endometrial cells originating from Day                   devoid of serum with PDBu (100 ng/ml). In experiments 7–9, samples of
14 cycling cows [17]. Systems used to evaluate secretion                     medium were collected at 6 h only. After collection, all samples were
of PGF2 from endometrial cells generally involve stimu-                      stored at 20 C until analyzed for PGF2 .
lation of cells with an activator of protein kinase C, such
as phorbol 12-myristate 13-acetate and phorbol 12,13 di-                     Preparation of Fatty Acid Solutions
butyrate (PDBu) [16]. These activators induce expression
of the PGHS-2 gene and stimulate secretion of PGF2 . The                         Stock solutions of polyunsaturated fatty acids were diluted in ethanol
                                                                             (50 mg/ml), aliquoted, and stored at 80 C. At preparation of culture
bIFN- attenuates both of these effects [15, 16].                             medium with fatty acid, the ethanol was evaporated with nitrogen gas, and
    The objectives of the present study were to determine                    the remaining concentrated fatty acid solution was diluted to 1 mM in
the effects of PUFAs on secretion of PGF2 from BEND                          culture medium (50% MEM and 50% Ham F-12) containing 33 mg/ml of
cells stimulated with PDBu, to test the interaction of                       fatty acid-free BSA (fraction V, low endotoxin). This solution was incu-
PUFAs with the inhibitory effect of bIFN- , and to explore                   bated for 2 h in a water bath at 37 C to allow binding of the fatty acid to
possible mechanisms of action by determining the effects                     BSA and then further diluted in culture medium to final treatment con-
                                                                             centrations. The BSA:fatty acid ratio was maintained constant at all fatty
of PUFAs on steady-state concentrations of PGHS-2                            acid concentrations tested.
mRNA and protein expression of PLA2 and PGHS-2.                                  Experiment 1. The objective of this experiment was to test the effect
                                                                             of different PUFAs and BSA on secretion of PGF2 induced by PDBu.
MATERIALS AND METHODS                                                        Cultures (3 ml of culture medium, six-well plates, n 30) were assigned
                                                                             randomly to incubation for 24 h with medium devoid of serum with no
Materials                                                                    fatty acid, medium devoid of serum with BSA (3.3 mg/ml), or medium
                                                                             devoid of serum with BSA and one of the following fatty acids (100 M):
    Recombinant bIFN- (1.08 107 U of antiviral activity per milligram)       EPA, DHA, AA, LNA, LA, or OA. Three 35-mm wells were assigned to
was donated kindly by Dr. R. Michael Roberts (University of Missouri,        each treatment. After the 24-h incubation, cells were washed with HBSS
Columbia, MO). Polystyrene tissue-culture Costar six-well plates and cul-    and exposed to culture medium containing PDBu (100 ng/ml). The control
ture dishes (100 20 mm) were purchased from Corning (Corning Glass           groups included cells incubated with BSA that subsequently were exposed
Works, Corning, NY). Polystyrene flasks for cell culture (175 cm2) were       or not exposed to PDBu and cells incubated for 24 h with culture medium
from Sarstedt, Inc. (Newton, NC). Acrylamide, N,N -methylenebisacry-         alone (no BSA) and subsequently stimulated with PDBu.
lamide, and Nonidet 40 were from BDH Laboratory Supplies (Poole,                 Experiment 2. This experiment was conducted to test the effect of
U.K.).                                                                       different concentrations of EPA, DHA, and LNA on PGF2 secretion from
    The Ham F-12, Eagle minimum essential medium (MEM), antibiotic-          BEND cells stimulated with PDBu. Cultures (3 ml of culture medium, six-
antimycotic solution (AbAm), 3-(N-Morpholino) propanesulfonic acid           well plates, n    54) were assigned in triplicate to incubation for 24 h in
(MOPS), and EDTA were from Sigma Chemical Co. (St. Louis, MO). The           medium devoid of serum with treatments of a 3 6 factorial experiment.
lysis buffer was from Promega (Reporter lysis buffer; Madison, WI). Iso-     Factors were fatty acid (EPA, DHA, or LNA) and concentration (0, 20,
topically labeled [5, 6, 8, 11, 12, 14, 15,-3H-PGF2 ] (212 Ci/mol), and      40, 60, 80, or 100 M). At 0 h, cells were washed twice with HBSS and
nitrocellulose membranes (Hybond-ECL) were from Amersham Pharmacia           treated with medium devoid of serum containing PDBu (100 ng/ml).
Biotech (Arlington Heights, IL). BioTrans Nylon membrane was from ICN            Experiment 3. This experiment was conducted to test the effect of
Chemicals (Irvine, CA). Hanks balanced salt solution (HBSS) and TRIzol       concentrations of EPA between 0 and 20 M on PGF2 secretion from
were from Life Technologies (Grand Island, NY). X-ray film was from           BEND cells stimulated with PDBu. Cultures (3 ml of culture medium, six-
Eastman Kodak Co. (X-Omat Blue XB-1; Rochester, NY). Enhanced                well plates, n 12) were assigned in triplicate to incubation with medium
chemiluminescence (ECL) kit (Renaissance Western Blot Chemilumines-          devoid of serum with BSA and 0, 6.25, 12.5, or 20 M EPA for 24 h and
cence Reagent Plus) was from NEN Life Science Products (Boston, MA).         subsequently washed and stimulated with PDBu (100 ng/ml) dissolved in
Anti-PGHS-2, PGHS-2 electrophoresis standard, and the fatty acids EPA        medium devoid of serum.
(C20:5, n-3), DHA (C22:6, n-3), AA (C20:4, n-6), linolenic (LNA; C18:            Experiment 4. This experiment was conducted to test the effect of
3, n-3), linoleic (LA; C18:2, n-6), and oleic (OA; 18:1, n-9) were pur-      different durations of incubation on PGF2 secretion from BEND cells
chased from Cayman Chemical (Ann Arbor, MI). Cytosolic PLA2 poly-            stimulated with PDBu. Cultures (3 ml of culture medium, six-well plates,
clonal antibody was purchased from Santa Cruz Biotechnologies, Inc.          n    18) were assigned in triplicate to incubation with medium devoid of
(Santa Cruz, CA). The bovine PGHS-2 cDNA probe was cloned from an            serum with BSA and 100 M EPA for 0, 1, 3, 6, 12, or 24 h. After
ovarian follicular cDNA library [18]. The probe was labeled by nick trans-   incubation, cells were washed and treated with PDBu (100 ng/ml) dis-
lation using a Nick Translation kit (N5000; Amersham Pharmacia Biotech,      solved in culture medium devoid of serum.
Little Chalfont, U.K.) and 32P-labeled deoxycytidine triphosphate (dCTP;         Experiment 5. This experiment was conducted to test the effect of
ICN Chemicals, Costa Mesa, CA). The procedure used has been described        concentrations of bIFN- between 0 and 25 ng/ml on PGF2 secretion from
by Badinga et al. [19].                                                      BEND cells stimulated with PDBu. Cultures (3 ml of culture medium, six-
                                                                             well plates, n      27) were incubated for 24 h with medium devoid of
Cell Culture and Experimental Designs                                        serum without fatty acids. After incubation, cells were washed and as-
                                                                             signed in triplicate to receive medium devoid of serum containing PDBu
    The BEND cell line is deposited and characterized by the American        (100 ng/ml) in combination with 0, 0.01, 0.05, 1, 2, 3.125, 12.5, or 25
Type Culture Collection (ARCC no. CRL-2398; Manassas, VA). The               ng/ml of bIFN- .
BEND cells (16,22) were plated on 35-mm wells of six-well plates (1              Experiment 6. This experiment was conducted to test the effect of
105 cells/well) in culture medium (40% Ham F-12, 40% MEM, 10 ml/L            combinations of different doses of EPA and bIFN- on secretion of PGF2
of AbAm, 200 U/L of insulin, 0.343 g/L of D-valine, 10% fetal bovine         from BEND cells stimulated with PDBu. Cultures (24 ml of culture me-
serum, and 10% horse serum) for experiments 1–6 and 9 or on 100-             dium, six-well plates, n      27) were assigned in triplicate to a 3      3
20-mm Petri dishes (2.4      106 cell/dish) for experiments 7 and 8. The     factorial experiment. Factors were incubated for 24 h with medium devoid
ratio of culture area to volume of culture medium was the same in both       of serum containing EPA (0, 3, or 20 M) and subsequent treatment with
culture containers. Cells were grown to confluence at 37 C in an incubator    bIFN- (0, 50, or 100 pg/ml) given concurrently with PDBu (100 ng/ml).
(model MCO 17AI; Sanyo Electric Co., Ltd., Osaka, Japan) under a hu-         At the end of incubation with EPA, cells were washed twice with HBSS
782                                                                MATTOS ET AL.

to remove EPA and BSA and treated with PDBu (100 ng/ml) dissolved             labeled with [32P]dCTP. The PGHS-2 mRNA transcript was identified by
in medium devoid of serum and containing the different concentrations of      autoradiography, and hybridization signals were analyzed by densitometry.
bIFN- . Whole-cell extracts (WCEs) were collected at 6 h to determine
protein yields. This experiment was replicated once.                          Radioimmunoassay
    Experiment 7. This experiment was conducted to compare the effect
of three concentrations bIFN- and three concentrations of EPA on PGF2             Concentrations of PGF2 were measured in medium samples collected
secretion and steady-state concentrations of PGHS-2 mRNA in BEND              at 0 and 6 h after treatment of cells with PDBu. Twenty-five microliters
cells stimulated with PDBu. Cultures (24 ml of culture medium, 100-mm         of each sample were further diluted in 75 l of 50 mM Tris-HCl (pH 7.5,
dishes, n     12) were assigned in duplicate to incubation for 24 h with      Tris buffer). Therefore, the total volume of diluted samples for assay was
medium devoid of serum containing 0, 20, or 100 M EPA followed by             100 l. Danet-Desnoyers et al. [14] have described the RIA procedure. To
treatment with 100 ng/ml of PDBu in serum-free medium without fatty           prepare standard curves, known amounts of nonradioactive PGF2 (1.25–
acids or incubation for 24 h with serum-free medium without fatty acids       1000 pg/tube) were diluted in Tris buffer (100 l). An anti-PGF2 anti-
followed by treatment at 0 h with 100 ng/ml of PDBu in combination with       serum [22] was diluted 1:5000 (Tris buffer, 100 l/tube). Final assay vol-
0, 0.5, or 50 ng/ml of bIFN- . Total RNA extraction was conducted at 6        ume was 400 l (100 l of Tris buffer, 100 l of diluted sample, 100 l
h. This experiment was replicated once.                                       of antibody solution, and 100 l of radiolabeled PGF2 solution). The
    Experiment 8. This experiment was conducted to test the effect of         minimum detectable concentration of PGF2 was 3.32 pg/tube. Inter- and
different concentrations of EPA, DHA, and AA on secretion of PGF2 and         intraassay coefficients of variation were 11.2% and 13.3%, respectively.
intracellular concentrations of PGHS-2 and cPLA2 protein in BEND cells        Assay was validated for serum-free medium by adding 25 pg of PGF2
stimulated with PDBu. Cultures (24 ml of culture medium, 100-mm dish-         per 0.1 ml to medium. Average recovered PGF2 was 25.1 1.42 pg per
es, n 12) were assigned in duplicate to incubation for 24 h with medium       0.1 ml, which yielded a calculated recovery of 100.25%. A test using
devoid of serum containing 100 M EPA, DHA, or AA. After incubation,           known amounts of PGF3 resulted in a cross-reactivity of 39.8%. Cross-
cells were washed twice with HBSS and treated with 100 ng/ml of PDBu          reactivity with PGE3 was less than 1%. Concentrations of PGF3 used in
dissolved in media devoid of serum. The WCEs were collected at 6 h after      the test of cross-reactivity were 0.625, 1.25, 2.5, 5, 10, and 20 ng/ml.
addition of PDBu for PGHS-2 and PLA2 immunoblotting. This experiment          Concentrations of PGE3 tested were 20, 40, 80, and 100 ng/ml. Concen-
was replicated once.                                                          trations of PGF2 are reported as picograms per milliliter of culture me-
    Experiment 9. This experiment was conducted to test the effect of         dium because treatments did not affect cell numbers, as determined by
combinations of EPA and AA concentrations on secretion of PGF2 from           protein yield (experiments 6 and 8).
BEND cells stimulated with PDBu. Cultures (3 ml of culture medium, six-
well plates, n    27) were assigned in triplicate to a 3   3 factorial ex-
periment. Cells were incubated for 24 h with medium devoid of serum
                                                                              Statistical Analyses
containing all factorial combinations of EPA (0, 25, and 100 M) and AA            Data were analyzed by least-squares ANOVA using the general linear
(0, 25, and 100 M). After incubation, cells were washed twice with            models procedure of SAS [23]. Data for each experiment were analyzed
HBSS and treated with 100 ng/ml of PDBu dissolved in medium devoid            separately. The PGF2 data were analyzed as a split-plot design. Models
of serum. Medium samples were at 6 h after PDBu.                              included the effects of treatment well (or dish) within treatment, time,
                                                                              interaction between time and treatment, and residual error. The effect of
                                                                              well (or dish) within treatment was used as the error term to test for the
Preparation of Cell Extracts and Immunoblotting Analysis                      effects of treatment. Preplanned orthogonal contrasts and pairwise com-
                                                                              parisons were used to identify differences between means. Concentrations
    The WCEs were harvested immediately after collection of the last me-
                                                                              of PGF2 in medium at 6 h after addition of PDBu were greater than at 3
dium sample. Cells were washed twice with HBSS at 4 C and incubated
                                                                              h (P     0.01), and the pattern of responses among treatments were com-
for 15 min with 1 ml of lysis buffer. Dishes were then scraped using a
                                                                              parable at 3 and 6 h. The 6-h responses for all experiments are reported.
rubber policeman to collect cell fragments attached to the dish. Lysates
                                                                              For abundance of PGHS-2 and cPLA2, the models included the effect of
and cell fragments were transferred to 1.5-ml conical tubes and agitated
                                                                              treatment only. Values of PGHS-2 mRNA abundance in experiment 7 were
for 5 min using a rotating device (Roto-torque; Cole-Parmer Instrument
                                                                              adjusted for variability of loading using the abundance of 18s rRNA as a
Co., Chicago, IL) to allow further cell lysis. Cell debris was separated by
                                                                              covariate. Preplanned pairwise comparisons were used to determine dif-
centrifugation using a microcentrifuge (13 000       g) for 2 min. Superna-
                                                                              ferences between means.
tants were transferred to 1.5-ml tubes and stored at 20 C. Concentrations
                                                                                  The test of homogeneity of polynomial regression [24] was used in
of protein in WCEs were determined using the Bradford method [21]. A
                                                                              experiment 2 to compare the inhibition curves obtained using increasing
sample of each WCE was diluted 1:25 (40 l of sample in 960 l of
                                                                              concentrations of EPA, DHA, and LNA.
ddH2O) and used for determination of protein concentration. Twenty-five
microgram of protein were loaded onto duplicate 7.5% acrylamide gels.
A PGHS-2 eletrophoresis standard (50 ng) also was loaded onto one lane        RESULTS
of the gel and used as a positive control. Proteins were separated by one-
dimensional SDS-PAGE (150 V, 100 mA, 25 min). Proteins were trans-            Effect of Polyunsaturated Fatty Acids on Secretion
ferred to nitrocellulose membranes as previously described [16]. After        of PGF2
transfer, membranes were blocked for 2 h in 5% nonfat dried milk and
subsequently incubated for 2 h with a polyclonal antibody against PGHS-          Incubation for 24 h with 100 M of the omega-3 fatty
2 (1:500 dilution in 5% nonfat dry milk solids in Tris-buffered saline        acids EPA, DHA, and LNA significantly attenuated secre-
[TBS]). After incubation with the primary antibody, membranes were            tion of PGF2 (P 0.05) in response to PDBu when com-
washed once with TBS containing 0.1% Tween 20 (TBST) for 15 min and           pared with incubation without fatty acid (Fig. 1). Arachi-
thrice for 5 min. Membranes were then incubated for 1 h with anti-rabbit      donic acid (100 M) had the opposite effect and tended to
immunoglobulin G horseradish peroxidase-linked whole antibody from
sheep (1:5000 dilution in TBST). Antibody complexes were detected using
                                                                              increase secretion of PGF2 in response to PDBu (P 0.1).
ECL, with exposure times of 1 min for PGHS-2 and 2 min for PLA2.              The other fatty acids tested (100 M), OA and LA, did not
Protein bands were quantified by densitometry as described by Binelli et       affect production of PGF2 (P        0.05). Incubation with
al. [16].                                                                     BSA without fatty acid did not affect secretion of PGF2 in
                                                                              response to treatment with PDBu. Cells incubated with
RNA Isolation and Northern Blot Analysis                                      BSA and not stimulated with PDBu did not secrete detect-
                                                                              able amounts of PGF2 .
   At 6 h, culture medium was aspirated and cells washed twice using             In experiment 2, incubation with increasing concentra-
HBSS at 4 C. Cells were lysed using 3 ml of Trizol at 4 C for 5 min.          tions (dose) of n-3 PUFAs resulted in a dose-responsive
Total RNA was extracted following the manufacturer’s instructions. The        reduction in secretion of PGF2 (P        0.001). Treatment
precipitated RNA was resuspended using 25 l of RNase-free water. Con-
centration of RNA was determined using a spectrophotometer set at
                                                                              with 20 M of any of the three fatty acids resulted in lower
OD260. Total cellular RNA (30 g/lane) was fractionated in a 1% agarose-       secretion of PGF2 in comparison to the control without
formaldehyde gel, stained with ethidium bromide, blotted to a nylon mem-      fatty acid (P    0.02). Treatment with EPA [Y  ˆ    6078
brane, and hybridized with a homologous bovine PGHS-2 cDNA probe              (225     dose)    (3.4     dose2)  (0.016     dose3); R2
                                        FATTY ACIDS REGULATE PROSTAGLANDIN SECRETION                                                                783




                                                                              FIG. 3. Experiment 4. Least-square means and SEM of concentrations of
                                                                              PGF2 in medium conditioned by BEND cells 6 h after treatment with
                                                                              PDBu (100 ng/ml). Cells were incubated with the fatty acid EPA (100 M)
                                                                              for 0 (Control), 1, 3, 6, 12, or 24 h before treatment with PDBu. Secretion
FIG. 1. Experiment 1. Least-square means and SEM of concentrations of
                                                                              of PGF2 in response to PDBu decreased with increasing incubation time
PGF2 in medium conditioned by BEND cells 6 h after treatment with
                                                                              (P     0.01). Differences between different incubation times and the Con-
PDBu (100 ng/ml). Cells were incubated with no fatty acid (Control) or
                                                                              trol: aP   0.1, *P     0.01, and **P     0.001.
with 100 M EPA, DHA, AA, LA, LNA, or OA for 24 h before treatment
with PDBu. A second control group was included to test the effect of BSA
used to solubilized the fatty acids. The BSA did not affect PGF2 secretion.
Differences between different fatty acids and the Control: aP     0.10, *P
   0.05; and **P     0.01.                                                    of the cells to the medium containing 100 M EPA (P
                                                                              0.01, experiment 4; Fig. 3).
                                                                                 Treatment of cells with PDBu in combination with in-
                   ˆ
0.92] and DHA [Y 5930 (212 dose) (3.1 dose2)                                  creasing concentrations of bIFN- (experiment 5) resulted
   (0.015     dose3); R2      0.95] resulted in clear dose re-                in a dose-dependent decrease in concentrations of PGF2
sponses and greater inhibition of PGF2 secretion than with                    (P      0.001). Concentrations of PGF2 in the medium of
LNA [Y  ˆ     6067      (96.6     dose)    (1.52     dose2)                   cells treated with bIFN- at 0.01, 0.1, 0.5, 1, 2, 3.125, 12.5,
(0.008 dose     3); R2    0.95; P 0.01] (Fig. 2). Treatment                   or 20 ng/ml were 6570, 4485, 3314, 2388, 2628, 1793, 966,
with 20 M LNA, DHA, or EPA resulted in a 22% (4835                            and 1131 pg/ml (SEM         398), respectively. Inhibition of
pg/ml, P     0.04), 60% (2464 pg/ml, P        0.01), and 61%                  PGF2 secretion was significant at all concentrations of
(2423 pg/ml, P       0.01) inhibition of PGF2 secretion at 6                  bIFN- tested when compared to the control without bIFN-
h in comparison to the control without fatty acid (6203 pg/                     (P     0.01).
ml, SEM       381), respectively. Results from experiment 2                      In Experiment 6, both incubation for 24 h with EPA (P
confirm the findings of experiment 1 for EPA, DHA, and                              0.002) and subsequent treatment with bIFN- (P
LNA. When tested at a lower dose range (experiment 3),                        0.001) inhibited secretion of PGF2 in response to PDBu
incubation for 24 h with EPA reduced secretion of PGF2                        (Fig. 4). No interaction between bIFN- and EPA was de-
in response to PDBu in a dose-dependent manner (P                             tected, indicating that EPA and bIFN- acted in an additive
0.01). Treatment with 6.25, 12.5, and 20 M EPA resulted                       manner. Treatments did not affect concentrations of protein
in an inhibition of PGF2 secretion at 6 h of 24.5% (3667                      measured in WCEs collected at 6 h.
pg/ml, P       0.02), 22.3% (3733 pg/ml, P          0.03), and
46.7% (2590 pg/ml, P          0.01), respectively, when com-
pared to the control without fatty acid (4859 pg/ml, SEM
   299).
   The inhibitory effect of EPA on secretion of PGF2 in-
duced by PDBu increased with increasing time of exposure




                                                                              FIG. 4. Experiment 6. Least-square means and SEM of concentrations of
                                                                              PGF2 in medium conditioned by BEND cells 6 h after treatment with
FIG. 2. Experiment 2. Prostaglandin F2 response curves for increasing         PDBu (100 ng/ml). Cells were assigned to a 3     3 factorial experiment.
concentrations (0, 20, 40, 60, 80, or 100 M) of the fatty acids EPA, DHA,     Factors were incubation for 24 h with 0, 3, or 20 M EPA and treatment
or LNA. Concentrations were measured in medium conditioned by BEND            with PDBu in combination with 0, 50 or 100 pg/ml of bIFN- . Both EPA
cells 6 h after treatment with PDBu (100 ng/ml). Cells were incubated for     and bIFN- attenuated the secretion of PGF2 in response to PDBu. Both
24 h before treatment with PDBu in a 3      6 factorial design. Both EPA      EPA and bIFN- inhibited secretion of PGF2 (P      0.01). The interaction
and DHA were more potent inhibitors than LNA (P         0.01).                between EPA and bIFN- was not significant.
784                                                MATTOS ET AL.

FIG. 5. Experiment 7. Northern blot anal-
ysis of PGHS-2 mRNA in BEND cells 6 h
after treatment with PDBu (100 ng/ml).
Cells were incubated with EPA (20 or 100
  M) for 24 h before treatment with PDBu
or were treated with PDBu in combination
with bIFN- (0.5 or 50 ng/ml). Controls
were treated with PDBu alone. Top) Auto-
radiographic exposure of abundance of
PGHS-2 mRNA (upper row) and ethidium
bromide staining of 18S ribosomal RNA
(lower row). Bottom) Least-square means
and SEM of abundance of PGHS-2 mRNA
arbitrary densitometric units. Details of
statistical differences are described in Re-
sults. The bIFN- , but not the EPA, attenu-
ated the increase in abundance of PGHS-2
mRNA induced by PDBu (P           0.01).




Effects of Polyunsaturated Fatty Acids and bIFN-             Effects of Polyunsaturated Fatty Acids
on Steady-State Concentrations of PGHS-2 mRNA:               on PGHS-2 and cPLA2 Protein Expression:
PGHS-2 Northern Blot Analysis                                PGHS-2 and PLA2 Immunoblotting
                                                                Concentrations of PGF2 in medium at 6 h after PDBu
   Incubation of cells with EPA for 24 h did not affect      were increased by incubation for 24 h with AA (1991
PGHS-2 mRNA concentrations (P 0.1). However, bIFN-           84.2 pg/ml, P     0.01) and decreased (P      0.01) by EPA
  reduced PGHS-2 mRNA concentrations in a dose-respon-       (567     84.2 pg/ml) and DHA (756      84.2 pg/ml) relative
sive manner (P      0.01, experiment 7; Fig. 5). Reduction   to the control with PDBu alone (1454 84.2 pg/ml). Con-
of PGHS-2 mRNA abundance relative to the control not         centrations of PGHS-2 were not affected by any of the fatty
treated with bIFN- was 26.5% and 90.7% for 0.5 and 50        acids tested (P     0.05). Treatment with DHA tended to
ng/ml of bIFN- , respectively. Both EPA and bIFN- atten-     reduce the abundance of PLA2 (P        0.08, experiment 8;
uated the secretion of PGF2 at 6 h after addition of PDBu    Fig. 6). Treatments did not affect concentrations of protein
(P 0.01). Concentrations of PGF2 in medium from con-         measured in WCEs collected at 6 h.
trols (no EPA and no bIFN- ) were greater (5504.3 217
pg/ml, P     0.01) than those in medium from cells treated   Effect of Coincubation with AA and EPA
with 20 M EPA (3100 307 pg/ml), 100 M EPA (1777              on PGF2 Secretion
   307 pg/ml), 0.5 ng/ml of bIFN- (2240        307 pg/ml),      Coincubation of BEND cells with AA and EPA for 24
or 50 ng/ml of bIFN- (334       307 pg/ml).                  h (experiment 9) demonstrated the competing effects of the


FIG. 6. Experiment 8. Immunoblot analy-
sis of PGHS-2 and PLA2 in whole-cell ex-
tracts collected from BEND cells 6 h after
treatment with PDBu (100 ng/ml). Cells
were incubated for 24 h before PDBu with
the fatty acids EPA (100 M), DHA (100
  M), or AA (100 M). a) Representative-
enhanced ECL exposure of abundance of
total PGHS-2 (top) and least-square means
   SEM of abundance of total PGHS-2 ar-
bitrary densitometric units (ADU; bottom).
b) Representative-enhanced ECL exposure
of abundance of total PLA2 (top) and least-
square means       SEM of abundance of to-
tal PGHS-2 ADU (bottom). Incubation
with DHA tended to decrease abundance
of PLA2. Std, Standard. *P    0.08.
                                      FATTY ACIDS REGULATE PROSTAGLANDIN SECRETION                                                  785

                                                                           alone. In other words, if EPA or other n-3 fatty acids indeed
                                                                           increased production of PGF3 , then the real differences in
                                                                           production of PGF2 between controls treated with PDBu
                                                                           alone and cells incubated with EPA or other n-3 fatty acids
                                                                           were greater than those detected by the RIA.
                                                                               Incubation of cells with culture medium containing a
                                                                           fatty acid bound to BSA increases the concentration of that
                                                                           particular fatty acid in the plasma membrane [25]. Exper-
                                                                           iment 4 demonstrated that the inhibitory effect of EPA on
                                                                           secretion of PGF2 increased with longer exposure of cells
                                                                           to EPA. Although incorporation of fatty acids to the plasma
                                                                           membrane was not measured in the present study, the mass
                                                                           of EPA incorporated to the plasma membrane likely in-
                                                                           creased with increasing incubation time, and this greater
                                                                           incorporation likely resulted in greater inhibition of PGF2
                                                                           secretion. It is possible that longer incubation periods
                                                                           would result in a greater inhibition of PGF2 secretion.
FIG. 7. Experiment 9. Least-squares means and SEM of concentrations        Therefore, it is also possible that the effects described in
of PGF2 in medium conditioned by BEND cells 6 h after treatment with       experiments 1–3 and 6–9 could have been attained using
PDBu (100 ng/ml). Cells were assigned to a 3     3 factorial experiment.   lower doses and longer incubation times.
Factors were incubated for 24 h with 0, 25, or 100 M EPA or AA. Se-            In experiments 1 and 2, incubation of BEND cells with
cretion of PGF2 in response to PDBu was attenuated by EPA (P       0.01)
and increased by AA (P     0.01). The interaction between EPA and AA
                                                                           the n-3 PUFAs EPA, DHA, and LNA caused a dose-de-
was not significant.                                                        pendent reduction of PGF2 secretion in response to stim-
                                                                           ulation with PDBu. Incubation for 24 h with EPA was sig-
                                                                           nificant even at the lowest concentration tested (6.25 M,
two fatty acids. Arachidonic acid increased (P 0.01) se-                   experiment 3). These findings are in agreement with reports
cretion of PGF2 in response to PDBu, whereas EPA was                       indicating that PUFAs of the n-3 and n-6 families can in-
inhibitory (P     0.01; Fig. 7). The response to incubation                hibit the secretion of prostanoids in several cell types cul-
with 25 M AA tended to decrease with increasing con-                       tured in vitro [7, 26]. Other studies have demonstrated re-
centrations of EPA (P        0.09, EPA      AA interaction).               duced prostanoid synthesis when fatty acids of the n-3 and
Incubation with 100 M EPA resulted in a 72.4% (2198                        n-6 families are fed in the diet [27]. It is not clear why
pg/ml) inhibition of PGF2 secretion relative to the untreat-               LNA was less inhibitory than DHA and EPA. Linolenic
ed control (7960 pg/ml), but this inhibitory effect was re-                acid is the precursor for synthesis of DHA and EPA and
versed with coincubation of EPA (100 M) with AA (100                       can be converted to them in a process that relies on activ-
  M), causing the concentrations of PGF2 in the medium                     ities of desaturase and elongase enzymes. It is possible that
to be similar to that of the control (6715 pg/ml, P 0.05).                 some conversion of LNA to EPA and DHA occurred during
                                                                           the period of incubation, leading to the observed reduction
DISCUSSION                                                                 in PGF2 secretion. The fact that LA did not affect secretion
   The experiments described indicate that PUFAs can                       of PGF2 in response to PDBu somewhat contradicts pre-
modify the secretion of PGF2 in a model in which BEND                      vious reports indicating an inhibitory activity of this fatty
cells are stimulated with PDBu. Binelli et al. [16] reported               acid [28, 29]. Moreover, LA has been considered to be a
that PDBu (100 ng/ml) increases steady-state concentra-                    potential mediator of reduced endometrial PGF2 secretion
tions of PGHS-2 mRNA and protein and induces secretion                     in the pregnant cow [1]. Conversely, because LA is the
of PGF2 . Moreover, it was determined that bIFN- (50 ng/                   most abundant precursor for synthesis of AA and PGF2 , it
ml) suppresses PGF2 , probably through reduction of                        could be hypothesized that LA would increase secretion of
PGHS-2 mRNA concentrations, protein expression, and en-                    PGF2 through increased precursor availability. This did not
zymatic activity of PGHS-2 and PLA2. A control without                     occur in the BEND cell system. Possible reasons could in-
PDBu was run only in experiment 1, because that experi-                    volve lack of an efficient system for conversion of LA to
ment demonstrated that incubation of cells with PUFA did                   AA, which involves two steps of desaturation and one step
not induce secretion of PGF2 . Concentrations of PGF2                      of elongation. The fact that incubation with 100 M AA
were undetectable in that group. In a previous report [16],                increased production of PGF2 (experiments 1 and 8) sup-
maximal induction of PGF2 secretion was detected be-                       ports the concept that availability of AA limits secretion of
tween 3 and 6 h after treatment of cells with PDBu and                     PGF2 and that conversion of LA to AA does not occur
coincided temporally with an increase in the intracellular                 efficiently in the cell culture system used. Oleic acid was
mass of PGHS-2 protein. Based on these results, the present                included as a fatty acid control that is not a substrate for
experiments were designed to determine the effects of fatty                the PGHS enzymes and does not interfere with synthesis
acids on secretion of PGF2 and enzyme expression (PGHS-                    of prostanoids [7]. Oleic acid did not affect secretion of
2 and PLA2) at 6 h after treatment with PDBu.                              PGF2 in response to stimulation with PDBu.
   The antibody used to determine concentrations of PGF2                       Experiment 6 demonstrated that EPA can amplify the
also had a significant cross-reactivity (39.8%) for PGF3 .                  inhibitory effects of bIFN- on secretion of PGF2 in an
Although we were unable to separate the contribution of                    additive manner. The concentrations of EPA and bIFN-
PGF3 to the PGF2 measurements, any increased produc-                       included in experiment 6 were based on the results of ex-
tion of PGF3 as a result of incubation with EPA or other                   periments 3 and 5, which determined concentrations of
n-3 fatty acids would have reduced the apparent differences                EPA and bIFN- that partially inhibit secretion of PGF2 .
of PGF2 concentrations measured by RIA between cells                       Utilizing these partially inhibitory doses, a significant am-
treated with PDBu and EPA and cells treated with PDBu                      plification in PGF2 inhibition was detected in experiment
786                                                             MATTOS ET AL.

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