- Email: [email protected]
5-Hydroxyeicosatetraenoic acid and human parturition
ELSEVIER
SHydroxyeicosatetraenoic Human Parturition S.S. EdwinA, R.J. RomeroB, M.D. Mitchell’
H. MunozB,
Acid and
D.W. BranchA and
*Department of Obstetrics and Gynecology, University of Utah BPerinatal Research Branch, National Institute of Health Child Health and Human Development ‘Department of Pharmacology and Clinical Pharmacology University of Auckland 5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonic acid (AA) metabolite derived from the lipoxygenase pathway which is capable of inducing uterine contractions. The purpose of this study was to determine a). whether 5-HETE concentrations in amniotic fluid increase before or after the onset of labor and b). whether acetylsalicylic acid (ASA) could modulate the production of 5-HETE by human amnion cells. 5-HETE concentrations are increased in amniotic fluid before the onset of labor. Furthermore, ASA treatment as expected inhibited PGE,, but also significantly increased 5-HETE production by amnion cells. 5-HETE concentrations on average increased by greater than 2.5 fold (p c 0.001) in amniotic fluid prior to spontaneous labor when compared with samples obtained from the same patients earlier in gestation and therefore may be important in mechanisms regulating the onset of labor. ASA provokes an increase in 5-HETE biosynthesis by amnion cells: control media 2.60 ? 1.5, ASA treatment alone 5.17 f 0.20, IL-I/3 alone 6.39 f 2.1, and ASA + IL-Z/3 8.95 -+ 1.2 (mean + SEM) picograms per microgram protein per 16 hours. These findings may explain in part why cyclooxygenase inhibitors are not always successful in treating women with preterm labor. Keywords: 5hydroxyeicosatetraenoic acetylsalicylic acid; preterm labor
acid; interleukin-113;
amniotic
fluid;
Correspondence and Reprint Requests to: Samuel S. Fdwin, Department of Obstetrics and Gynecology, University of Utah School of Medicine, Salt Lake City, Utah, Phone: (801) 581-7167, FAX: (801) 581-7199 Prostaglandins 51:403-412, 1996 0 1996 by Elsevier Science Inc. 65.5 Avenue of the Americas, New York, NY 10010
0090-6980/96/$15.00 PII SOO90-6980(96)00046-9
Acetylsalicylic Acid and 5HETE Biosynthesis: Edwin et al. Introduction Mobilization of free arachidonic acid (AA) from glycerophospholipids of the fetal membranes is thought to be a key step leading to the initiation of human labor’. AA is metabolized principally through 3 pathways: 1). the cyclooxygenase pathway leading to the formation of prostaglandins and thromboxanes, 2). the epoxygenase pathway leading to the formation of epoxides, and 3). the lipoxygenase pathway leading to the formation of hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (LTs). Uterine tissues have relatively higher amounts of arachidonic acid in comparison to other tissues in the human body and release of AA from fetal membranes alone is thought to be sufficient to account for all of the prostaglandins formed during the onset of labor’. Although prostaglandins are thought to be key mediators in the initiation of labor, a growing body of evidence suggests that arachidonate lipoxygenase products, particularly SHETE, may play an important role in term and preterm labor. Fetal membranes, decidua, and the placenta can synthesize 5-HETE’ which is capable of causing concentration dependent contractions of the human myometrium3. It is thought that concentrations of arachidonic acid metabolites with uterine contractile properties may increase in amniotic fluid prior to the onset of labor in preparation for labor. Therefore, in preparation for the onset of spontaneous labor amniotic fluid concentrations of 5-HETE may also increase prior to the onset of labor. An increase in amniotic fluid 5-HETE concentration could modulate uterine contractility in both term and preterm labor especially in situations where Non-steroidal anti-inflammatory drugs (NSAIDs) have been used to treat preterm labor presumably by inhibiting prostaglandin production. Concentrations of inflammatory cytokines including interleukin- 113(IL18) are increased in amniotic fluid of women in preterm labor with intraamniotic infection4.(j. We have shown that inflammatory cytokines stimulate 5HETE production by gestational tissues’. Also, concentrations of arachidonate lipoxygenase products including 5-HETE are increased in the amniotic fluid of women with preterm labor with intraamniotic infection *-lo. Therefore, increased 5-HETE concentrations in the amniotic fluid may reflect physiologic process which could play a key role in the mechanisms regulating the onset of both preterm and term labor. The purpose of this study was two fold: (1) to determine if amniotic fluid 5-HETE concentrations are elevated prior to the onset of labor, and (2) to ascertain the effects of NSAIDs such as aspirin on the regulation 5-HETE production by human amnion.
404
Prostaglandins
1996: 5 1, June
Acetylsalicylic Acid and 5-HETE Biosynthesis: Edwin et al.
Materials and Methods Amniotic Fluids Amniotic fluid was obtained by transabdominal amniocentesis in 21 women beginning at the 37th week of gestation. Informed consents were obtained from all patients. Amniocenteses were performed under ultrasound guidance for lung maturity, and to search for meconium in patients with intrahepatic cholestasis. All amniocenteses were performed prior to the spontaneous onset of labor. Amniotic fluid samples were centrifuged at 200 g for 10 minutes at 4°C and stored in polypropylene tubes at -20°C until assayed.
Primary Cell Cultures Placentae were obtained from 4 individual women at scheduled repeat cesarean deliveries at term. All patients had normal pregnancies without evidence of intraamniotic infection and were not in labor. Immediately following delivery, the amnion was peeled off the chorionic membranes, and the decidua was sharply dissected off the chorion. Amnion was minced and incubated with trypsin (1.2% in DMEM) for 2 hours at 37°C as previously described”. Following the digestion, cells were washed thoroughly and cultured in Ham’s F12fDME media (Irvine Scientific, CA) supplemented with 10% fetal calf serum (Gibco, NY) in 24 well tissue culture plates (Costar, Van Nuys, CA). Primary cultures of amnion cells were grown to confluence (5-7 days) prior to experimentation.
Experimental
Conditions
Experiments were performed in quadruplicate on confluent amnion cells for 16 hours in humidified air containing 5% CO, in the presence and absence of IL- 113 (1 ng/ml) or control media. Additionally, amnion cells were also incubated with IL-la in the presence and absence of ASA (25 200 PM). IL- 18 was purchased from R&D Systems, Minneapolis, MN. Acetylsalicylic acid was purchased from Sigma chemical company, St. Louis, MO. Confluent cells were rinsed three times with serum free medium and all experiments were conducted in the absence of calf serum. Following incubation, supernatants were collected and stored frozen at -20°C.
Assays 5-HETE and PGE2 were assayed directly in the culture media by specific radioimmunoassays8,9”2”3. The antisera for each metabolite were purchased from PerSeptive BioResearch Products Inc., Cambridge, MA
Prostaglandins 1996: 5 1, June
405
Acetylsalicylic Acid and 5HETE
Biosynthesis: Edwin et al.
and have negligible cross reactivity with the experimental conditions. An equivalent volume of non-conditioned medium was added to the standards in the standard curves. Cellular proteins were solubilized and measured using the method of Lowry et a1,14. 5-HETE was extracted from amniotic fluid samples using five volumes of acidified diethyl ether. The organic solvent was evaporated under a steady stream of nitrogen and the residue dissolved in phosphate buffer (pH 7.4) prior to assaying for 5HETE. The extraction efficiency was calculated to be 71.28%. All amniotic fluid samples were run in a single assay in duplicate and solvent blanks were subtracted from measured values. Statistics Statistical differences were assessed by using Wilcoxon matched paired test and two-way ANOVA. Experimental conditions in quadruplicate were tested on at least 4 individual placentae. A p value of less than 0.05 was considered significant. When this difference was not achieved it is designated as p = NS.
Red ts Our initial studies focused on the actions of acetylsalicylic acid on human amnion cell arachidonate 5lipoxygenase metabolite formation, particularly 5-HETE. When arachidonic acid metabolism through the cyclooxygenase pathway is inhibited by treatment with NSAIDs such as aspirin, AA continues to be metabolized through the lipoxygenase pathway resulting in the formation of uterine contractile agents such as 5-HETE [figure 11. As expected, PGE, production by amnion cells was drastically reduced by aspirin treatment. Surprisingly, we detected a massive increase in 5-HETE production in amnion cells treated with varying concentrations of aspirin (control vs aspirin at 200 PM; p < 0.05) [figure 21. We then focused our efforts to determine the regulation of 5-HETE production by amnion cells in response to aspirin. For this purpose we evaluated both 5-HETE and PGE, production by amnion cells stimulated with IL-ll3 in the presence of ASA. IL-la was chosen since our experience suggests that it is the most potent and consistent stimulator of prostaglandin production in cultured human gestational tissues. We measured 5-HETE, and PGE, production by amnion cells in the presence and absence of IL-lf3 (1 ng/ml) with and without aspirin (200 PM) treatment. As expected, both 5-HETE and PGE, production was significantly elevated (p c 0.001) in the presence of IL-lI3 (1 ng/ml) in these cells. PGE, production by IL-H3 stimulated amnion cells treated with aspirin was drastically diminished (IL-lf3 vs IL-la + aspirin; p < 0.001) whereas 5-HETE production (IL- 113vs IL-lI3 + aspirin; p = NS) remained unchanged [Figure 31.
406
Prostaglandins
1996: 5 1, June
Acetylsalicylic Acid and 5-HETE Biosynthesis: Edwin et al.
GLYCEROPHOSPHOLIPIDS
Phospholiposes
+ ARACHIDONIC
ACID
,’
‘, ‘*\
5-HE
TE
I’ ,’
OK
5-HETE
FIGURE 1. A simplifiedschematic representationof !?I-HETE and PGE, formation through the lipoxygenase and cyclooxygenase pathways in the presence and absence of aspirin, a NSAID like drug.
We also evaluated 5HETE concentrations in serial amniotic fluids of patients (n = 21) just prior to the onset of labor [figure 41. 5HETE concentrations were significantly elevated in the amniotic fluid obtained during the second amniocentesis when compared with amniotic fluid from the first amniocentesis. A mean increase greater than 2.5 fold (p < 0.001) was detected in samples obtained just prior to the onset of labor as compared to samples obtained from the same patients earlier in gestation.
Discussion Arachidonic acid metabolites, particularly the cyclooxygenase metabolites produced by intrauterine tissues, have been studied extensively over the past 10 years. However, little is known about arachidonate lipoxygenase metabolite production by human gestational tissues. Among the most
Prostaglandins 1996: 5 1, June
407
Acetylsalicylic Acid and 5HETE Biosynthesis: Edwin et al. -5
E ‘rl
, -
PGE,
Y u
lII.l
-
0
Ii
0
I cl
I 25
I 50
Aspirin
I 200
concentration
( uM )
FIGURE2. The effects of varying concentrations of aspirin (acetylsalicylic acid) on amnion cell 5HETE and PGE, production (mean f SEM, n = 4). For 5-HETE and PGE,, control vs aspirin : p < 0.001 (aspirin at 200 ~JM).
biologically active products of the lipoxygenase pathway are 5-hydroxyeicosatetraenoic acid (SHETE), 12hydroxyeicosatetraenoic acid (12HETE), 1 Shydroxyeicosatetraenoic acid (1 SHETE), leukotriene B4 (LTB4), and leukotriene C4 (LTC4). Currently, there is a growing body of evidence suggesting an important role for arachidonate lipoxygenase metabolites particularly 5-HETE and LTC4, in the mechanisms regulating the onset of 1aboP. Rhesus monkeys treated with indomethacin can deliver with no increase in amniotic fluid prostaglandin concentration but with increased In addition, studies in monkeys production of lipoxygenase metaboliteP. during late pregnancy showed significant changes in amniotic fluid concentrations of lipoxygenase products, particularly 5-HETE, occurring prior to spontaneous delivery. Similarly, in situ studies with guinea pig uterus suggest possible involvement of lipoxygenase products in myometrial contractionP. We have shown that human intrauterine tissues are capable of producing arachidonate lipoxygenase metabolites including 5-HETE”,‘*, and it is known that 5-HETE can cross the fetal Furthermore, in studies with human myometrial strips membranes”.
408
Prostaglandins 1996: 5 1, June
Acetylsalicylic Acid and 5-HETE Biosynthesis: Edwin et al.
6000
E
\
5000
z ;
4000
0 Is
b
2
3000
a, ii
i3
2000
0
1
1st Amniocentesis
2nd
Amniocente&
FIGURE4. The amniotic fluid 5-HETE concentrations in women prior to the onset of labor. Amniotic fluid samples obtained at least a week prior to the onset of labor (first amniocentesis) vs samples obtained just prior to the onset of labor (second amniocentesis): p < 0.001.
tissues. This phenomenon could be one explanation for the inability of cyclooxygenase inhibitors such as indomethacin to curtail preterm labor in all patients. We speculate that the products of AA metabolism through the lipoxygenase pathway and the products of AA metabolism through the cyclooxygenase pathway may act in concert to initiate the process of labor. Although prostaglandins are thought to be the universal mediators of labor, the importance of arachidonate lipoxygenase metabolites including 5HETE should be considered as another potential biochemical mechanism in the genesis and propagation of uterine contractions of human labor.
References 1.
410
Okita, J.R., MacDonald, P.C., Johnston, J.M. Mobilization of arachidonic acid from specific glycerophospholipids of human fetal membranes during early labor. J Biol Chem 252: 14029-34. 1982.
Prostaglandins 1996: 5 1, June
Acetylsalicylic Acid and 5-HETE Biosynthesis:
LA.
Edwin et al
”
Control
Aspirin
IL- 15 + Aspirin
Aspirin
IL- lp
.Y
4
v
s
*-
3
+.J
U
-z
2
0 a”
1 0 Control
IL- I!3
f
Aspirin
FIGURE 3. The effects of aspirin on BHETE and PGE, production (mean f SEM, n = 4) by amnion cells. For 5HETE, control vs IL-l&: p < 0.001 (IL-IR at 1 nglml), control vs aspirin: p < 0.05, and IL-l R vs IL-l I3 + aspirin: P = NS. For PGE2, control vs IL-l R: p < 0.001, control vs aspirin: p c 0.05, and IL-l I3vs IL-113+ aspirin: p c 0.001.
obtained before labor it was shown that S-HETE stimulated contractions in a dose dependent marine?‘‘’ suggestive of a potentially direct role for this arachidonate lipoxygenase metabolite in eliciting human uterine contractions. All of these studies suggest an important role for 5-HETE in the cascade of biochemical events leading up to the onset of labor. Our data are the first to show that amniotic fluid 5-HETE concentration increases prior to the onset of labor in humans. Since 5-HETE is capable of inducing uterine contractility, it is possible that the mechanisms underlying labor also include 5-HETE in addition to the more recognized prostaglandins. Our data on amnion cell 5-HETE production in the presence of aspirin, a NSAID like drug which inhibits the biosynthesis of prostaglandins, adds credence to the importance of 5 HETE as an important mediator of labor. Our data also show that aspirin like drugs can stimulate intrauterine tissues to release arachidonate lipoxygenase metabolite formation (such as 5-HETE) while simultaneously inhibiting prostaglandin biosynthesis by these same
ProstagZundins 1996: 5 1, June
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Acetylsalicylic Acid and 5HETE Biosynthesis: Edwin et al 2.
3
4.
5
6.
7
8.
9.
10.
11.
12.
13. 14. 15.
16. 17.
Saeed, S.A., Mitchell, M.D. Formation of arachidonate lipoxygenase metabolites by human fetal membranes, uterine decidua Vera and placenta. Prostaglandins Leukotrienes Med &635-40. 1981. Bennet, P.R., Murdoch, G.E., Myatt, L. The effects of lipoxygenase metabolites of arachidonic acid on human myometrial contractility. Prostaglandins %:83744. 1987. Romero, R., Brody, D.T., Qyarzun, E., Mazor, M., Wu, Y.K., Hobbins, J.C., Duran, S.K. Infection and labor. III. Interleukin-1: a signal for the onset of parturition. Am. J. Obstet. Gynecol. m(5 Pt 1):1117-23. 1989. Romero, R., Mazor, M., Sepulveda, W., Avila, C., Copeland, D., Williams, J. Tumor necrosis factor in preterm and term labor. Am. J. Obstet. Gynecol. m(5):1576-87. 1992. Romero R, Avila C, Santhanam U, Sehgal PB. Amniotic fluid interleukin-6 in preterm labor, association with infection. Journal of Clinical Investigation x:1392-400. 1990. Edwin, S.S., LaMarche, S.L., Thai, D., Branch, M.D., Mitchell, M.D. 5Hydroxyeicosatetraenoic acid biosynthesis by gestational tissues: Effects of inflammatorycytokines. Am. J. Obstet. Gynecol. m:1467-71. 1993. Romero, R., Wu, Y.K., Mazor, M., Hobbins, J.C., Mitchell, M.D. Amniotic fluid 5-hydroxyeicosatetraenoic acid in preterm labor. Prostaglandins 36: 179-89. 1988. Romero, R., Quintero, R., Emamian, M., Wan, M., Grzyboski, C., Hobbins, J.C., Mitchell, M.D. Arachidonate lipoxygenase metabolites in amniotic fluid of women with intraamniotic infection and preterm labor. Am. J. Obstet. Gynecol. m:1454-60. 1987. Romero, R., Wu, Y.K., Mazor, M., Oyarzun, E., Hobbins, J.C., Mitchell, M.D. Amniotic fluid arachidonate lipoxygenase metabolites in preterm labor. Prostaglandins Leukotrienes EFA &?69-75. 1989. Okita, J.R., Sagawa, N., Casey, M.L., Sneider, J.M. Comparison of human amnion tissue and amnion cells in primary culture by morphological and biochemical criteria. In Vitro. 19: 117-26. 1983. Lundin-Schiller, S., Mitchell, M.D. Regulation of chorion laeve prostaglandin E, production by epidermal growth factor, protein kinase C activation and calcium. Placenta u:597-03. 1991. Mitchell, M.D. The regulation of decidual prostaglandin biosynthesis by growth factors, phorbol esters, and calcium. Biol. Reprod &&:87 l-74. 199 1. Lowry OH, Rosebrough NF, Farr AL, Randall RJ. Protein measurements with the Folin reagent. J Biol Chem m:265-75. 1951. Walsh, SW. Evidence for 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C4 (LTC4) in the onset of labor. Ann. N.Y. Acad. Sci. m:341-54. 1991. Carraher, R., Hahn, D.W., Ritchie, D.M., McGuire, J.L. Involvement of lipoxygenase products in myometrial contractions. Prostaglandins %(1):23-32. 1983. Edwin, S.S., Mitchell, M.D. Arachidonate lipoxygenase metabolite formation in gestational tissues. J. Lipid Mediators Cell Signalling 9:291-300. 1991.
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Acetylsalicylic Acid and 5HETE Biosynthesis: Edwin et al. 18.
19.
20.
Editor:
412
Edwin, S.S., Thai, D., LaMarche, S., Branch, D.W., Mitchell, M.D. The regulation of arachidonate lipoxygenase metabolite formation in cells derived from intrauterine tissues. Prostaglandins Leukotrienes and EFA a:229-33. 1995. Bennett, P.R., Chamberlain, G.V., Pate& L., Elder, M.G., Myatt, L. Mechanisms of parturition: the transfer of prostaglandin E, and 5hydroxyeicosatetraenoic acid across fetal membranes. Am. J. Obstet. Gynecol. m:683-87. 1990. Bennett, P.R., Elder, M.G., Myatt, L. The effects of hpoxygenase metabolites of arachidonic acid on human myometrial contractility. Prostaglandins %:837-44. 1987.
M. Bygdeman
Received:
07-28-95
Accepted:
03- 12-96
ProstagZandins 1996: 5 1, June
SHydroxyeicosatetraenoic Human Parturition S.S. EdwinA, R.J. RomeroB, M.D. Mitchell’
H. MunozB,
Acid and
D.W. BranchA and
*Department of Obstetrics and Gynecology, University of Utah BPerinatal Research Branch, National Institute of Health Child Health and Human Development ‘Department of Pharmacology and Clinical Pharmacology University of Auckland 5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonic acid (AA) metabolite derived from the lipoxygenase pathway which is capable of inducing uterine contractions. The purpose of this study was to determine a). whether 5-HETE concentrations in amniotic fluid increase before or after the onset of labor and b). whether acetylsalicylic acid (ASA) could modulate the production of 5-HETE by human amnion cells. 5-HETE concentrations are increased in amniotic fluid before the onset of labor. Furthermore, ASA treatment as expected inhibited PGE,, but also significantly increased 5-HETE production by amnion cells. 5-HETE concentrations on average increased by greater than 2.5 fold (p c 0.001) in amniotic fluid prior to spontaneous labor when compared with samples obtained from the same patients earlier in gestation and therefore may be important in mechanisms regulating the onset of labor. ASA provokes an increase in 5-HETE biosynthesis by amnion cells: control media 2.60 ? 1.5, ASA treatment alone 5.17 f 0.20, IL-I/3 alone 6.39 f 2.1, and ASA + IL-Z/3 8.95 -+ 1.2 (mean + SEM) picograms per microgram protein per 16 hours. These findings may explain in part why cyclooxygenase inhibitors are not always successful in treating women with preterm labor. Keywords: 5hydroxyeicosatetraenoic acetylsalicylic acid; preterm labor
acid; interleukin-113;
amniotic
fluid;
Correspondence and Reprint Requests to: Samuel S. Fdwin, Department of Obstetrics and Gynecology, University of Utah School of Medicine, Salt Lake City, Utah, Phone: (801) 581-7167, FAX: (801) 581-7199 Prostaglandins 51:403-412, 1996 0 1996 by Elsevier Science Inc. 65.5 Avenue of the Americas, New York, NY 10010
0090-6980/96/$15.00 PII SOO90-6980(96)00046-9
Acetylsalicylic Acid and 5HETE Biosynthesis: Edwin et al. Introduction Mobilization of free arachidonic acid (AA) from glycerophospholipids of the fetal membranes is thought to be a key step leading to the initiation of human labor’. AA is metabolized principally through 3 pathways: 1). the cyclooxygenase pathway leading to the formation of prostaglandins and thromboxanes, 2). the epoxygenase pathway leading to the formation of epoxides, and 3). the lipoxygenase pathway leading to the formation of hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (LTs). Uterine tissues have relatively higher amounts of arachidonic acid in comparison to other tissues in the human body and release of AA from fetal membranes alone is thought to be sufficient to account for all of the prostaglandins formed during the onset of labor’. Although prostaglandins are thought to be key mediators in the initiation of labor, a growing body of evidence suggests that arachidonate lipoxygenase products, particularly SHETE, may play an important role in term and preterm labor. Fetal membranes, decidua, and the placenta can synthesize 5-HETE’ which is capable of causing concentration dependent contractions of the human myometrium3. It is thought that concentrations of arachidonic acid metabolites with uterine contractile properties may increase in amniotic fluid prior to the onset of labor in preparation for labor. Therefore, in preparation for the onset of spontaneous labor amniotic fluid concentrations of 5-HETE may also increase prior to the onset of labor. An increase in amniotic fluid 5-HETE concentration could modulate uterine contractility in both term and preterm labor especially in situations where Non-steroidal anti-inflammatory drugs (NSAIDs) have been used to treat preterm labor presumably by inhibiting prostaglandin production. Concentrations of inflammatory cytokines including interleukin- 113(IL18) are increased in amniotic fluid of women in preterm labor with intraamniotic infection4.(j. We have shown that inflammatory cytokines stimulate 5HETE production by gestational tissues’. Also, concentrations of arachidonate lipoxygenase products including 5-HETE are increased in the amniotic fluid of women with preterm labor with intraamniotic infection *-lo. Therefore, increased 5-HETE concentrations in the amniotic fluid may reflect physiologic process which could play a key role in the mechanisms regulating the onset of both preterm and term labor. The purpose of this study was two fold: (1) to determine if amniotic fluid 5-HETE concentrations are elevated prior to the onset of labor, and (2) to ascertain the effects of NSAIDs such as aspirin on the regulation 5-HETE production by human amnion.
404
Prostaglandins
1996: 5 1, June
Acetylsalicylic Acid and 5-HETE Biosynthesis: Edwin et al.
Materials and Methods Amniotic Fluids Amniotic fluid was obtained by transabdominal amniocentesis in 21 women beginning at the 37th week of gestation. Informed consents were obtained from all patients. Amniocenteses were performed under ultrasound guidance for lung maturity, and to search for meconium in patients with intrahepatic cholestasis. All amniocenteses were performed prior to the spontaneous onset of labor. Amniotic fluid samples were centrifuged at 200 g for 10 minutes at 4°C and stored in polypropylene tubes at -20°C until assayed.
Primary Cell Cultures Placentae were obtained from 4 individual women at scheduled repeat cesarean deliveries at term. All patients had normal pregnancies without evidence of intraamniotic infection and were not in labor. Immediately following delivery, the amnion was peeled off the chorionic membranes, and the decidua was sharply dissected off the chorion. Amnion was minced and incubated with trypsin (1.2% in DMEM) for 2 hours at 37°C as previously described”. Following the digestion, cells were washed thoroughly and cultured in Ham’s F12fDME media (Irvine Scientific, CA) supplemented with 10% fetal calf serum (Gibco, NY) in 24 well tissue culture plates (Costar, Van Nuys, CA). Primary cultures of amnion cells were grown to confluence (5-7 days) prior to experimentation.
Experimental
Conditions
Experiments were performed in quadruplicate on confluent amnion cells for 16 hours in humidified air containing 5% CO, in the presence and absence of IL- 113 (1 ng/ml) or control media. Additionally, amnion cells were also incubated with IL-la in the presence and absence of ASA (25 200 PM). IL- 18 was purchased from R&D Systems, Minneapolis, MN. Acetylsalicylic acid was purchased from Sigma chemical company, St. Louis, MO. Confluent cells were rinsed three times with serum free medium and all experiments were conducted in the absence of calf serum. Following incubation, supernatants were collected and stored frozen at -20°C.
Assays 5-HETE and PGE2 were assayed directly in the culture media by specific radioimmunoassays8,9”2”3. The antisera for each metabolite were purchased from PerSeptive BioResearch Products Inc., Cambridge, MA
Prostaglandins 1996: 5 1, June
405
Acetylsalicylic Acid and 5HETE
Biosynthesis: Edwin et al.
and have negligible cross reactivity with the experimental conditions. An equivalent volume of non-conditioned medium was added to the standards in the standard curves. Cellular proteins were solubilized and measured using the method of Lowry et a1,14. 5-HETE was extracted from amniotic fluid samples using five volumes of acidified diethyl ether. The organic solvent was evaporated under a steady stream of nitrogen and the residue dissolved in phosphate buffer (pH 7.4) prior to assaying for 5HETE. The extraction efficiency was calculated to be 71.28%. All amniotic fluid samples were run in a single assay in duplicate and solvent blanks were subtracted from measured values. Statistics Statistical differences were assessed by using Wilcoxon matched paired test and two-way ANOVA. Experimental conditions in quadruplicate were tested on at least 4 individual placentae. A p value of less than 0.05 was considered significant. When this difference was not achieved it is designated as p = NS.
Red ts Our initial studies focused on the actions of acetylsalicylic acid on human amnion cell arachidonate 5lipoxygenase metabolite formation, particularly 5-HETE. When arachidonic acid metabolism through the cyclooxygenase pathway is inhibited by treatment with NSAIDs such as aspirin, AA continues to be metabolized through the lipoxygenase pathway resulting in the formation of uterine contractile agents such as 5-HETE [figure 11. As expected, PGE, production by amnion cells was drastically reduced by aspirin treatment. Surprisingly, we detected a massive increase in 5-HETE production in amnion cells treated with varying concentrations of aspirin (control vs aspirin at 200 PM; p < 0.05) [figure 21. We then focused our efforts to determine the regulation of 5-HETE production by amnion cells in response to aspirin. For this purpose we evaluated both 5-HETE and PGE, production by amnion cells stimulated with IL-ll3 in the presence of ASA. IL-la was chosen since our experience suggests that it is the most potent and consistent stimulator of prostaglandin production in cultured human gestational tissues. We measured 5-HETE, and PGE, production by amnion cells in the presence and absence of IL-lf3 (1 ng/ml) with and without aspirin (200 PM) treatment. As expected, both 5-HETE and PGE, production was significantly elevated (p c 0.001) in the presence of IL-lI3 (1 ng/ml) in these cells. PGE, production by IL-H3 stimulated amnion cells treated with aspirin was drastically diminished (IL-lf3 vs IL-la + aspirin; p < 0.001) whereas 5-HETE production (IL- 113vs IL-lI3 + aspirin; p = NS) remained unchanged [Figure 31.
406
Prostaglandins
1996: 5 1, June
Acetylsalicylic Acid and 5-HETE Biosynthesis: Edwin et al.
GLYCEROPHOSPHOLIPIDS
Phospholiposes
+ ARACHIDONIC
ACID
,’
‘, ‘*\
5-HE
TE
I’ ,’
OK
5-HETE
FIGURE 1. A simplifiedschematic representationof !?I-HETE and PGE, formation through the lipoxygenase and cyclooxygenase pathways in the presence and absence of aspirin, a NSAID like drug.
We also evaluated 5HETE concentrations in serial amniotic fluids of patients (n = 21) just prior to the onset of labor [figure 41. 5HETE concentrations were significantly elevated in the amniotic fluid obtained during the second amniocentesis when compared with amniotic fluid from the first amniocentesis. A mean increase greater than 2.5 fold (p < 0.001) was detected in samples obtained just prior to the onset of labor as compared to samples obtained from the same patients earlier in gestation.
Discussion Arachidonic acid metabolites, particularly the cyclooxygenase metabolites produced by intrauterine tissues, have been studied extensively over the past 10 years. However, little is known about arachidonate lipoxygenase metabolite production by human gestational tissues. Among the most
Prostaglandins 1996: 5 1, June
407
Acetylsalicylic Acid and 5HETE Biosynthesis: Edwin et al. -5
E ‘rl
, -
PGE,
Y u
lII.l
-
0
Ii
0
I cl
I 25
I 50
Aspirin
I 200
concentration
( uM )
FIGURE2. The effects of varying concentrations of aspirin (acetylsalicylic acid) on amnion cell 5HETE and PGE, production (mean f SEM, n = 4). For 5-HETE and PGE,, control vs aspirin : p < 0.001 (aspirin at 200 ~JM).
biologically active products of the lipoxygenase pathway are 5-hydroxyeicosatetraenoic acid (SHETE), 12hydroxyeicosatetraenoic acid (12HETE), 1 Shydroxyeicosatetraenoic acid (1 SHETE), leukotriene B4 (LTB4), and leukotriene C4 (LTC4). Currently, there is a growing body of evidence suggesting an important role for arachidonate lipoxygenase metabolites particularly 5-HETE and LTC4, in the mechanisms regulating the onset of 1aboP. Rhesus monkeys treated with indomethacin can deliver with no increase in amniotic fluid prostaglandin concentration but with increased In addition, studies in monkeys production of lipoxygenase metaboliteP. during late pregnancy showed significant changes in amniotic fluid concentrations of lipoxygenase products, particularly 5-HETE, occurring prior to spontaneous delivery. Similarly, in situ studies with guinea pig uterus suggest possible involvement of lipoxygenase products in myometrial contractionP. We have shown that human intrauterine tissues are capable of producing arachidonate lipoxygenase metabolites including 5-HETE”,‘*, and it is known that 5-HETE can cross the fetal Furthermore, in studies with human myometrial strips membranes”.
408
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Acetylsalicylic Acid and 5-HETE Biosynthesis: Edwin et al.
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4000
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FIGURE4. The amniotic fluid 5-HETE concentrations in women prior to the onset of labor. Amniotic fluid samples obtained at least a week prior to the onset of labor (first amniocentesis) vs samples obtained just prior to the onset of labor (second amniocentesis): p < 0.001.
tissues. This phenomenon could be one explanation for the inability of cyclooxygenase inhibitors such as indomethacin to curtail preterm labor in all patients. We speculate that the products of AA metabolism through the lipoxygenase pathway and the products of AA metabolism through the cyclooxygenase pathway may act in concert to initiate the process of labor. Although prostaglandins are thought to be the universal mediators of labor, the importance of arachidonate lipoxygenase metabolites including 5HETE should be considered as another potential biochemical mechanism in the genesis and propagation of uterine contractions of human labor.
References 1.
410
Okita, J.R., MacDonald, P.C., Johnston, J.M. Mobilization of arachidonic acid from specific glycerophospholipids of human fetal membranes during early labor. J Biol Chem 252: 14029-34. 1982.
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Acetylsalicylic Acid and 5-HETE Biosynthesis:
LA.
Edwin et al
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IL- 15 + Aspirin
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FIGURE 3. The effects of aspirin on BHETE and PGE, production (mean f SEM, n = 4) by amnion cells. For 5HETE, control vs IL-l&: p < 0.001 (IL-IR at 1 nglml), control vs aspirin: p < 0.05, and IL-l R vs IL-l I3 + aspirin: P = NS. For PGE2, control vs IL-l R: p < 0.001, control vs aspirin: p c 0.05, and IL-l I3vs IL-113+ aspirin: p c 0.001.
obtained before labor it was shown that S-HETE stimulated contractions in a dose dependent marine?‘‘’ suggestive of a potentially direct role for this arachidonate lipoxygenase metabolite in eliciting human uterine contractions. All of these studies suggest an important role for 5-HETE in the cascade of biochemical events leading up to the onset of labor. Our data are the first to show that amniotic fluid 5-HETE concentration increases prior to the onset of labor in humans. Since 5-HETE is capable of inducing uterine contractility, it is possible that the mechanisms underlying labor also include 5-HETE in addition to the more recognized prostaglandins. Our data on amnion cell 5-HETE production in the presence of aspirin, a NSAID like drug which inhibits the biosynthesis of prostaglandins, adds credence to the importance of 5 HETE as an important mediator of labor. Our data also show that aspirin like drugs can stimulate intrauterine tissues to release arachidonate lipoxygenase metabolite formation (such as 5-HETE) while simultaneously inhibiting prostaglandin biosynthesis by these same
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Acetylsalicylic Acid and 5HETE Biosynthesis: Edwin et al 2.
3
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Saeed, S.A., Mitchell, M.D. Formation of arachidonate lipoxygenase metabolites by human fetal membranes, uterine decidua Vera and placenta. Prostaglandins Leukotrienes Med &635-40. 1981. Bennet, P.R., Murdoch, G.E., Myatt, L. The effects of lipoxygenase metabolites of arachidonic acid on human myometrial contractility. Prostaglandins %:83744. 1987. Romero, R., Brody, D.T., Qyarzun, E., Mazor, M., Wu, Y.K., Hobbins, J.C., Duran, S.K. Infection and labor. III. Interleukin-1: a signal for the onset of parturition. Am. J. Obstet. Gynecol. m(5 Pt 1):1117-23. 1989. Romero, R., Mazor, M., Sepulveda, W., Avila, C., Copeland, D., Williams, J. Tumor necrosis factor in preterm and term labor. Am. J. Obstet. Gynecol. m(5):1576-87. 1992. Romero R, Avila C, Santhanam U, Sehgal PB. Amniotic fluid interleukin-6 in preterm labor, association with infection. Journal of Clinical Investigation x:1392-400. 1990. Edwin, S.S., LaMarche, S.L., Thai, D., Branch, M.D., Mitchell, M.D. 5Hydroxyeicosatetraenoic acid biosynthesis by gestational tissues: Effects of inflammatorycytokines. Am. J. Obstet. Gynecol. m:1467-71. 1993. Romero, R., Wu, Y.K., Mazor, M., Hobbins, J.C., Mitchell, M.D. Amniotic fluid 5-hydroxyeicosatetraenoic acid in preterm labor. Prostaglandins 36: 179-89. 1988. Romero, R., Quintero, R., Emamian, M., Wan, M., Grzyboski, C., Hobbins, J.C., Mitchell, M.D. Arachidonate lipoxygenase metabolites in amniotic fluid of women with intraamniotic infection and preterm labor. Am. J. Obstet. Gynecol. m:1454-60. 1987. Romero, R., Wu, Y.K., Mazor, M., Oyarzun, E., Hobbins, J.C., Mitchell, M.D. Amniotic fluid arachidonate lipoxygenase metabolites in preterm labor. Prostaglandins Leukotrienes EFA &?69-75. 1989. Okita, J.R., Sagawa, N., Casey, M.L., Sneider, J.M. Comparison of human amnion tissue and amnion cells in primary culture by morphological and biochemical criteria. In Vitro. 19: 117-26. 1983. Lundin-Schiller, S., Mitchell, M.D. Regulation of chorion laeve prostaglandin E, production by epidermal growth factor, protein kinase C activation and calcium. Placenta u:597-03. 1991. Mitchell, M.D. The regulation of decidual prostaglandin biosynthesis by growth factors, phorbol esters, and calcium. Biol. Reprod &&:87 l-74. 199 1. Lowry OH, Rosebrough NF, Farr AL, Randall RJ. Protein measurements with the Folin reagent. J Biol Chem m:265-75. 1951. Walsh, SW. Evidence for 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C4 (LTC4) in the onset of labor. Ann. N.Y. Acad. Sci. m:341-54. 1991. Carraher, R., Hahn, D.W., Ritchie, D.M., McGuire, J.L. Involvement of lipoxygenase products in myometrial contractions. Prostaglandins %(1):23-32. 1983. Edwin, S.S., Mitchell, M.D. Arachidonate lipoxygenase metabolite formation in gestational tissues. J. Lipid Mediators Cell Signalling 9:291-300. 1991.
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Edwin, S.S., Thai, D., LaMarche, S., Branch, D.W., Mitchell, M.D. The regulation of arachidonate lipoxygenase metabolite formation in cells derived from intrauterine tissues. Prostaglandins Leukotrienes and EFA a:229-33. 1995. Bennett, P.R., Chamberlain, G.V., Pate& L., Elder, M.G., Myatt, L. Mechanisms of parturition: the transfer of prostaglandin E, and 5hydroxyeicosatetraenoic acid across fetal membranes. Am. J. Obstet. Gynecol. m:683-87. 1990. Bennett, P.R., Elder, M.G., Myatt, L. The effects of hpoxygenase metabolites of arachidonic acid on human myometrial contractility. Prostaglandins %:837-44. 1987.
M. Bygdeman
Received:
07-28-95
Accepted:
03- 12-96
ProstagZandins 1996: 5 1, June