Epidemiological studies and interventional clinical trials indicate that consumption of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) such as docosahexaenoic acid (DHA) lengthen gestational duration. Although the mechanisms are not well understood, prostaglandins (PG) of the 2-series are known to play a role in the initiation and progress of labor. In animal studies, modest DHA provision has been shown to reduce placental and uterine PGE2 and PGF2α, matrix metalloproteinase (MMP)-2 and MMP-9 expression, and placental collagenase activity. However, modulation of PG biosynthesis may not account for all the effects of LC n-3 PUFAs in labor. We investigated one potential PG-independent mechanism of LC PUFA action using cultured pregnant human myometrial smooth muscle cells. Our goal was to characterize the effect of LC PUFA treatment on oxytocin signaling, a potent uterotonic hormone involved in labor. The addition of 10 µM–100 µM DHA or arachidonic acid (AA) to the culture media for 48 h resulted in dose dependent enrichment of these fatty acids in membrane lipid. DHA and AA significantly inhibited phosphatidylinositol turnover and [Ca2+]i mobilization with oxytocin stimulation compared to bovine serum albumin control and equimolar oleic acid. DHA and AA significantly reduced oxytocin receptor membrane concentration without altering binding affinity or rate of receptor internalization. These findings demonstrate a role for LC n-3 PUFAs in regulation of oxytocin signaling and provide new insight into additional mechanisms pertaining to reports of dietary fish and fish oil consumption prolonging gestation.
Citation: Kim PY, Zhong M, Kim Y-S, Sanborn BM, Allen KGD (2012) Long Chain Polyunsaturated Fatty Acids Alter Oxytocin Signaling and Receptor Density in Cultured Pregnant Human Myometrial Smooth Muscle Cells. PLoS ONE 7(7): e41708. https://doi.org/10.1371/journal.pone.0041708
Editor: Kang Sun, Fudan University, China
Received: January 30, 2012; Accepted: June 25, 2012; Published: July 26, 2012
Copyright: © Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Colorado Agriculture Experiment Station Grant NC 1167 and NIH HD09618. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The Centers for Disease Control reports that the percentage of infants delivered preterm (<37 weeks of completed gestation) increased sharply from 9.4% in 1981 to 12% in 2010. The percentage of infants born low birth weight (<2500 g) rose from 6.6% to 8.15% during this period . Analysis of gestational duration from 1992 to 2002 has shown a shift in median duration from 40 weeks in 1992 to 39 weeks in 2002, which was accompanied by marked significant decreases in deliveries at ≥40 weeks and increases in those at 34 to 39 weeks . Shortened gestation and low birth weight are major risk factors for death and morbidity in infants –. While medical advances have improved preterm infant outcomes, efforts to prevent preterm birth have been much less successful.
Evidence from observational and clinical studies indicate that essential fatty acids of the n-3 and n-6 series may potentially modify gestational duration . Early epidemiological studies in Denmark by Olsen and colleagues found a significant increase in gestational duration with increasing maternal erythrocyte long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) , . In subsequent randomized control trials, LC n-3 PUFA supplementation during pregnancy has been shown to increase gestational duration by 4 to 6 days –. These findings have recently been replicated by others ,  and are supported by a recent systematic review and meta-analysis . The mechanism remains largely unknown.
Evidence indicates that prostaglandins (PGs) play a key role in initiating and maintaining normal labor . PGE2 and PGF2α increase at term in laboring women compared with nonlaboring women , , can activate tissue remodeling matrix metalloproteinases –, mediate cervical ripening , , and induce uterine contractions , . These PG2-mediated actions coupled with markedly elevated arachidonic acid (AA) in maternal erythrocytes of women delivering prematurely , apparent LC n-3 PUFA deficiency in these women , and the opposing effects of LC n-3 PUFAs and AA on PG2 biosynthesis – have pointed investigators toward a mechanism in which LC n-3 PUFAs prolong pregnancy by suppressing the AA cascade , , . Elevated reproductive tissue cytokine and PG production have been implicated in preterm birth involving intrauterine infection , but the role of PGs in idiopathic preterm labor is less well defined. In the absence of infection, PG concentrations in chorion, decidua, and myometrium are reported to be lower in women delivering prematurely compared with women delivering at term –. Although the threshold for effectiveness has not been established, these findings suggest that mechanisms other than or in addition to PG suppression may account for the observed increase in gestational duration with LC n-3 PUFA supplementation in expectant women .
Independent of PG biosynthesis, LC n-3 PUFAs have been shown to exert their cardioprotective  and immunosuppressive ,  effects at the level of the plasma membrane. Docosahexanoic acid (DHA)-induced changes in the membrane structure and architecture leading to modulation of ion channels and the displacement of key proteins from lipid raft signaling platforms appear to be the mechanisms of action –. Manipulation of the lipid environment has also been shown to modulate the action of oxytocin –, a potent uterotonic hormone involved in labor . Binding of oxytocin to its plasma membrane receptor activates phospholipase C to produce inositol 1,4,5-trisphosphate (IP3) from the hydrolysis of phosphatidylinositol bisphosphate. Activation of the IP3 receptor releases Ca2+ from intracellular stores, resulting in an increase in the concentration of the calcium-calmodulin complex. In smooth muscle, this complex activates myosin light chain kinase, which phosphorylates the 20 kDa light chain of myosin and facilitates contraction. In humans, myometrial sensitivity to oxytocin is significantly increased in women delivering preterm and decreased in women delivering post-term .
To complement the observations of lengthened gestation with DHA and fish oil supplementation and demonstrate one potential PG-independent mechanism, we examined the effect of LC PUFA on oxytocin signaling in pregnant human myometrial cells. The goal of our study was to characterize changes in the cell membrane lipid composition in response to the addition of micromolar concentrations of the free fatty acids oleic (OA), AA, and DHA into the culture media and to determine whether incorporation of these exogenous fatty acids into the myometrial plasma membrane affects oxytocin-mediated signaling events involved in smooth muscle contraction.
Materials and Methods
Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), trypsin, and other cell culture reagents were acquired from HyClone (Logan, UT). Fatty acid-free bovine serum albumin (BSA) was obtained from Sigma-Aldrich (St. Louis, MO). Fatty acids and fatty acid methyl ester standards were from Nu-Chek Prep (Elysian, MN). Oxytocin and other compounds were from Sigma-Aldrich. [3H]-myoinositol and [3H]-oxytocin were from Perkin Elmer New England Nuclear (Waltham, MA).
Immortalized pregnant human myometrial cells (PHM1-41) retain many morphological and phenotypic responses of primary myometrial cells, express smooth muscle-specific α-actin, and retain oxytocin receptors –. PHM1-41 cells were maintained in DMEM supplemented with 10% FBS and 2 mM L-glutamine in a humidified atmosphere of 5% CO2 at 37°C. Cells used in the study were between passages 20–25.
Fatty acid supplementation
Stock solutions of the fatty acids 18∶1 oleic acid (OA), 20∶4 arachidonic acid (AA), and 22∶6 docosahexanoic acid (DHA) were prepared in absolute ethanol at a concentration of 0.5 M, purged in argon gas, and stored at −20°C. 100 µM fatty acid media was prepared by adding fatty acid free BSA to cell culture media to a final BSA concentration of 50 µM, then incubating overnight with 2 µl of 0.5 M fatty acid stock solution per 10 ml media. 30 µM and 10 µM fatty acid media were prepared by dilution of 100 µM fatty acid media with BSA-free media. BSA-only media served as our control. The final ethanol concentration was adjusted to 0.02%. Fatty acid composition in the final media was confirmed by gas chromatography (see below). For all experiments, the cells were placed in fatty acid or control media 48 h after subculture; cells were grown for an additional 48 h and the treatment media were completely removed prior to assays.
Cell membrane preparation
PHM1-41 cells were seeded at a density of 5.5×105 cells/dish in 100 mm tissue culture dishes for 4 days. After rinsing twice with cold phosphate buffered saline (PBS), cells were harvested in ice-cold hypotonic buffer (20 mM HEPES, 10 mM EDTA, pH 7.4) as described previously . Cells were mechanically disrupted by 4×15 sec 25,000-rpm pulses with an Ultra Turrax T8 homogenizer at 4°C. Cell homogenates were centrifuged at 500× g for 10 min at 4°C and the post-nuclear supernatants were spun at 27,000× g for 30 min at 4°C. The resulting crude membrane pellets were suspended in 20 mM HEPES buffer (pH 7.4) and stored at −80°C until use.
Fatty acid extraction and analysis
Lipids were extracted from membrane pellets by chloroform∶methanol (2∶1 v/v) according to the method of Folch et al . Methyl esters were prepared by 14% boron trifluoride methanolysis according to the method of Morrison and Smith . Fatty acid composition was determined by gas chromatography on an Agilent 6890 Series GC (Santa Clara, CA) equipped with a flame ionization detector and a J&W Scientific DB-225 (30 m×0.25 mm I.D., 0.15 µm) capillary column (Folsom, CA). The oven temperature was ramped from an initial temperature of 100°C to 167°C at a rate of 10.65°C/min, then raised from 167°C to 206°C at a rate of 2.58°C/min and held for 8 min. Fatty acids were identified by matching retention times with known standards.
Intracellular Ca2+ measurement
PHM1-41 cells were plated at a density of 8×104 cells/dish in 35 mm uncoated glass-bottom dishes (MatTek, Ashland MA). After 4 days in culture, the cells were washed three times with fluorescence buffer (145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 0.5 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 5 mM glucose, pH 7.4) and loaded with 5 µM of Fura-2-acetoxymethylester (Fura-2 AM, Invitrogen, Carlsbad, CA) for 30 min. Cells were then washed three times with fluorescence buffer and allowed to hydrolyze Fura-2 AM for 30 min. After a final set of washes in fluorescence buffer, 20–25 individual cells were analyzed for changes in intracellular free calcium [Ca2+]i in response to 25 nM oxytocin. [Ca2+]i was measured using an InCyt2 Im2 imaging system with excitation at 340 nm and 380 nm and emission at 510 nm (Intracellular Imaging, Cincinnati OH). Data are expressed as the average peak increase in [Ca2+]i per dish.
PHM1-41 cells were seeded at a density of 1.2×105 cells/well in six-well tissue culture plates (Corning, Acton, MA). After 3 days, cells were labeled with 0.4 µM [3H]-myoinositol in serum-free DMEM overnight. The cells were washed twice with PBS then incubated in 1 ml of HBSS containing 10 mM LiCl and 0.1% BSA for 30 min at 37°C. Cells were treated with 100 nM oxytocin in HBSS or HBSS vehicle. Following 30 min incubation at 37°C, the media was aspirated and 1 ml of 20 mM cold formic acid was added. The cells were incubated for 30 min at 4°C then the acid was neutralized with 370 ul of 150 mM NH4OH. [3H]-inositol phosphates (IP, IP2, IP3) were isolated from cell lysates by ion exchange chromatography using AG1-X8 resin (100–200 mesh formate form, Bio-Rad, Hercules, CA) and increasing concentrations of ammonium formate in 0.1 N formic acid as described previously . Data are expressed as agonist dependent activity.
Oxytocin receptor binding assay
Total cell membrane pellets were quickly thawed at 37°C and centrifuged at 20,000× g for 30 min at 4°C. Membrane pellets were resuspended in membrane buffer (50 mM Tris, 10 mM MgCl2, pH 7.4) by homogenizing on ice. [tyrosyl-2,6-3H]-oxytocin was diluted in ligand buffer (50 mM Tris, 0.1% BSA, 10 mM MgCl2, pH 7.4). 96-well Multiscreen FB filter plates (Millipore, Bedford, MA) were pre-wet with 100 µl of membrane buffer for 30 min. The wetting buffer was removed by vacuum filtration and 100 µl of membrane sample was added to each well along with 100 µl of appropriate concentrations of [tyrosyl-2,6-3H]-oxytocin. Plates were incubated on a reciprocal shaker for 2 hr at room temperature. The binding reaction was stopped by rapid filtration under vacuum to separate bound ligand from free ligand. The plates were washed twice with 200 µl of cold wash buffer (50 mM Tris, 0.1% BSA, pH 7.4). The filters containing the bound ligand were punched out and total binding was determined by liquid scintillation counting (Scintisafe Econo 1 in Packard Tri-carb 2900TR). Nonspecific binding was determined by carrying out the binding with 10 µM of unlabeled oxytocin in the reaction mixture. The level of specific binding was calculated as the difference between total and nonspecific levels of binding. Oxytocin receptor maximal binding capacity (Bmax) and affinity (Kd) were estimated using a one-site binding model (GraphPad Software, San Diego CA).
Oxytocin receptor internalization assay
PHM1-41 cells were seeded into six-well tissue culture plates (Corning) at ∼1.2×105 cells/well. After 4 days in culture, cells were washed twice with chilled PBS and incubated for 3 hr at 4°C in 1 mL of Ca2+ free HBSS containing 5 mM MgCl2, 0.1% BSA, and 10 nM [tyrosyl-2,6-3H]-oxytocin. Following 3 hr equilibration at 4°C, the cells were incubated at 37°C for specific time points, the HBSS was removed, the cells were washed twice with chilled HBSS (Ca2+ free, Mg2+ free, 0.1% BSA), then washed with 400 µL of membrane wash solution (150 mM NaCl, 50 mM acetic acid, pH 2.8) to collect membrane bound receptors. To collect internalized receptors, cells were next lysed with 400 µL of 1 M HCl and neutralized with 400 µL of 1 M NaOH. Total binding was determined by liquid scintillation counting (Scintisafe Econo 1 in Packard Tri-carb 2900TR). Nonspecific binding wells contained an additional 1 µM of unlabeled oxytocin. The level of specific binding was calculated as the difference between total and nonspecific levels of binding. Internalized receptors at each incubation time were expressed as a percentage of total receptors (specific counts in cell lysate/total of specific membrane-bound and cell lysate counts).
Fatty acid incorporation into membrane
Initial experiments determined that there was no difference in membrane FA composition of cells grown in DMEM containing 10% serum with or without the addition of FA-free BSA. The addition of exogenous fatty acids to the growth media resulted in incorporation and enrichment of that particular fatty acid in the membrane lipid of PHM1-41 cells (Figure 1). Supplementing cells with 10 µM AA resulted in a small, but nonsignificant, increase in membrane AA as a proportion of total fatty acids as compared to equimolar BSA supplementation. With 10 µM DHA supplementation there was a marked increase in membrane DHA because endogenous DHA is very low in these cells under normal culture conditions. Increasing the concentration of exogenous AA or DHA to 30 µM and 100 µM significantly enriched the respective fatty acid in the membrane as compared to equimolar BSA supplementation. OA is among the most common membrane fatty acids in PHM1-41 cells (and the FBS in the growth media); therefore significant enrichment with OA supplementation was not observed at concentrations below 100 µM. The incorporation of exogenous fatty acids appeared to be dose dependent, as membrane lipids were more enriched at 100 µM supplementation than at 10 µM supplementation.
Membrane (A) 18∶1 OA, (B) 20∶4 AA, (C) 22∶6 DHA are shown as a percent of total membrane fatty acids. Values are mean percent ± SEM (n = 3 experiments). Bars not sharing common letters are significantly different between treatment groups, p<0.05.
An increase in the relative proportion of one fatty acid was accompanied by a decrease in the relative proportion of one or more other fatty acids. The relative increases in membrane AA or DHA in PUFA supplemented cells came at the expense of endogenous membrane OA. Increases in membrane 16∶0 palmitic acid and 18∶0 stearic acid also contributed to the decrease in membrane OA in PUFA supplemented cells (data not shown). OA accounted for 30±2.7% of total membrane fatty acids in cells grown in DMEM +10% FBS. In comparison, OA accounted for 18.2±1.6% and 16.7±1.6% of total membrane fatty acids in 30 µM AA and DHA treated cells and 15.9±0.2% and 16.4±0.3% in 100 µM AA and DHA treated cells, respectively. There is no significant difference in membrane OA between 30 µM and 100 µM PUFA treated cells, suggesting that the differences in oxytocin signaling observed between these two concentrations are not due to depletion of OA, but rather due to enrichment of AA or DHA in the membrane.
Myometrial cell intracellular Ca2+
Oxytocin has been previously shown to rapidly increase [Ca2+]i in PHM1-41 and primary myometrial cells . We assessed the effect of the changes in membrane composition of PHM1-41 cells by measuring transient increases in [Ca2+]i in response to 25 nM oxytocin and 100 nM thapsigargin stimulation (Figure 2). Thapsigargin releases [Ca2+]i by inhibiting sarco/endoplasmic reticulum intracellular Ca2+-ATPase pumps. No significant differences were found in thapsigargin-induced [Ca2+]i release between treatment groups. There were no significant differences in oxytocin-induced [Ca2+]i mobilization between BSA and OA treatments. In contrast, oxytocin-induced [Ca2+]i mobilization was significantly decreased 39% and 42% with 100 µM AA and 57% and 60% with 100 µM DHA treatments compared to equimolar BSA or OA treatments, respectively (Figure 3A).
Cells were treated with (A) 50 µM BSA, (B) 100 µM OA, (C) 100 µM AA, (D) 100 µM DHA. Arrows indicate application of 25 nM oxytocin (solid) and 100 nM thapsigargin (dashed). Values are mean of 23–26 cells.
The response of all cells are shown in (A) and cells unresponsive to oxytocin stimulation are excluded from the analysis in (B). Data are expressed as the mean peak increase in [Ca2+]i per dish. Values are mean ± SEM (n = 7 experiments at 10 µM and 30 µM; n = 12 experiments at 100 µM). Bars not sharing common letters are significantly different between treatment groups, p<0.05.
Almost all cells cultured in BSA that were selected for imaging responded to oxytocin with an increase in [Ca2+]i. However, we observed considerable heterogeneity in the response of PUFA treated cells, i.e., some cells in the dish responded robustly, some weakly, and some not at all. When individual cells that exhibited no response to oxytocin were excluded from the analysis, the effect of 100 µM DHA treatment was less pronounced, but still represented a significant decrease of 25% compared to equimolar BSA or OA treatments, whereas 100 µM AA treatment was not significantly different (Figure 3B). Thus, the decrease in oxytocin-induced [Ca2+]i mobilization observed with 100 µM PUFA could be partially attributed to a decrease in the proportion of oxytocin-sensitive cells as well as to attenuated oxytocin-mediated Ca2+ signaling in responsive cells.
Since oxytocin can mobilize [Ca2+]i as a consequence of production of the second messenger IP3 , we evaluated the effect of membrane lipid composition on phosphatidylinositol (PI) turnover (Figure 4). Treatment with 10 µM fatty acids had no effect on total [3H]-inositol phosphates generated in response to 25 nM oxytocin. Treatment with 30 µM PUFA significantly decreased PI turnover compared to equimolar OA, but not BSA (AA, p = 0.12; DHA, p = 0.11) treatment. Treatment with 100 µM AA and 100 µM DHA significantly decreased PI turnover by 53% and 50% respectively as compared to equimolar BSA treatment. There were no significant differences between BSA and OA treatment at any concentration.
Oxytocin receptor ligand binding and internalization
Treatment with 10 µM PUFA had no effect on the number of oxytocin receptor binding sites (Bmax). Treatment with 30 µM and 100 µM PUFA significantly decreased Bmax in a dose dependent manner without affecting affinity (Kd) compared to equimolar BSA or OA treatments (Table 1). There were no significant differences in Bmax or Kd between BSA and OA treatment at any concentration. The Bmax (3–4 pmol/mg protein) and Kd (≈1 nM) of controls were within the range of previously published values for the oxytocin receptor in human term myometrium , . Treatment with PUFAs did not significantly alter the internalization of the oxytocin receptor (Figure 5). There was less ligand bound overall in 100 µM AA and 100 µM DHA treated cells (data not shown), reflecting the reduced number of oxytocin receptor binding sites, but the rate of internalization between 0 min and 30 min time points did not change compared to equimolar BSA treated cells. Maximum internalization was observed between 30 min and 60 min time points.
Data are expressed as a percent of total cell-associated ligand (membrane bound+internalized). Values are mean ± SEM (n = 3 experiments). Points not sharing common letters are significantly different between treatment groups, p<0.05.
To our knowledge, the present study is the first to apply LC PUFAs to the culture medium of human myometrial myocytes. Various investigators have incubated isolated cardiomyocytes , , vascular myocytes , and colonocytes  using LC n-3 PUFAs bound to albumin at concentrations ranging from 5 µM to 214 µM and exposure durations ranging from 24 h to 96 h. Our treatment regimen of 10 µM to 100 µM fatty acid incubation for 48 h falls within the range of these previous reports and we detected no impact on cell viability or morphology at these low micromolar concentrations. In the last trimester of pregnancy, maternal plasma free (non-esterified) fatty acid values of AA and DHA are 16 µM and 15 µM, respectively . Most of the human plasma AA and DHA reside in esterified forms, mainly as phospholipid constituents of lipoproteins . This phospholipid pool is generally overlooked in terms of fatty acid transport. Nevertheless, the uptake and hydrolysis of liproproteins such as LDL would act as an in vivo delivery mechanism for not only cholesterol but also for the fatty acids of the LDL particle, contributing to the amount of fatty acids transported in the free pool. Thus the concentrations employed in the present study are within the physiological range.
Several studies have implicated eicosanoids in the regulation of gestational duration, parturition and the initiation of labor , , , . Both PG and leukotrienes (LT), such as PGE2, PGF2α, LTC4, and LTB4 increase prior to the onset of labor. Exogenous PGE2 or PGF2α provision induces cervical ripening and uterine contractions in both full-term and preterm labor –. Selective inhibitors of cyclooxygenase-2 are effective in decreasing PGE2 in amniotic tissues and have been explored for preventing preterm deliveries, although there are many complicating issues. PGE2 levels in amnion, chorion, decidua and myometrium from women in labor, whether preterm or term, were 2- to 5- fold higher than those in samples from nonlaboring women . However, PGE2 levels in women delivering preterm, whether in labor or not in labor, were markedly lower than either laboring or nonlaboring women delivered at term . Another study of PG concentrations in labor has reported that both PGE2 and PGF2α levels were significantly lower in amnion and placenta in women delivered preterm than in women delivered at term .
Considering these observations, it is difficult to reconcile the inhibitory effect of LC n-3 PUFAs on PG biosynthesis ,  as the sole mechanism of action for DHA in prolonging gestation. Besides their effects on production of PGs, LC n-3 PUFAs can act directly at the plasma membrane level. This was first demonstrated by Leaf et al regarding the antiarrhythmic effect of fish and fish oil consumption . The precise site of action in cardiomyocytes remains to be fully elucidated, but data from Anderson and colleagues  suggest that LC n-3 PUFAs may electrically stabilize cardiomyocytes by modulating ion channels through changes in membrane fluidity and elasticity in the microdomain bordering the transmembrane protein.
LC n-3 PUFAs have also been shown to exert their anti-inflammatory properties through eicosanoid-independent membrane effects. The successful application of LC n-3 PUFAs in the treatment of immune-inflammatory diseases is based largely on their ability to suppress T lymphocyte activation , . A number of key proteins involved in signal transduction are known to associate with lipid rafts, targeted there by acylation with saturated fatty acids . LC n-3 PUFAs have been proposed to disrupt T cell activation by displacing raft-resident signaling proteins such as Src family kinases and transmembrane linker for activation of T cells from lipid rafts , . LC n-3 PUFAs are thought to act primarily by altering raft composition and structure, although changes in protein fatty acylation may contribute to the displacement of proteins from lipid rafts . These observations raise the possibility that dietary LC n-3 PUFAs prolong gestational duration through changes in the lipid bilayer properties and associated changes in signaling mechanisms.
In the present study we demonstrated that both AA and DHA significantly alter the membrane lipid composition of PHM1-41 pregnant human myometrial smooth muscle cells (Figure 1). We are not aware of any fish oil supplementation trials in pregnant women that have characterized myometrial membrane fatty acid composition. Data for maternal erythrocyte phospholipids does exist for an approximate comparison. Dunstan et al have reported increases in maternal erythrocyte DHA as a percentage of total fatty acids at 37 weeks gestation, from 6.6±0.2% in the control group to 11.0±0.2% with fish oil consumption equivalent to one fatty fish meal per day (total of 2.2 g DHA and 1.1 g EPA per day) . In the present study, PHM1-41 membrane DHA composed 6.7±0.5% of total fatty acids with 30 µM DHA treatment and 10.2±0.5% with 100 µM DHA treatment. Relative proportions of other fatty acids were similar between erythrocytes from women fed fish oil and DHA treated PHM1-41 cells. Stearic acid and OA each composed 14–16% of total fatty acids in both erythrocytes and in myocytes treated with 30 µM and 100 µM DHA.
The increase in membrane AA and DHA was associated with suppressed oxytocin-stimulated [Ca2+]i mobilization without affecting the size of the releasable thapsigargin-sensitive [Ca2+]i store (Figures 2 and 3). Since the cells were washed several times during the Ca2+ dye loading procedure, an interpretation based on a direct effect of free fatty acid as proposed in cardiomyocytes and rat aortic smooth muscle cells  appears unlikely. Inhibition of the oxytocin-stimulated rise in [Ca2+]i was associated with near abolition of PI turnover (7.9±9.7% and 14.2±8.8% over basal for 100 µM AA and 100 µM DHA treated cells, respectively) (Figure 4).
Upstream of PI turnover, we found that 100 µM LC PUFA reduced the number of oxytocin receptor binding sites in the membrane without affecting binding affinity (Table 1). Fahrenholz et al have demonstrated that membrane cholesterol acts as an allosteric modulator of the oxytocin receptor , . High-affinity state receptors are stabilized three-fold (t1/2) and enriched two-fold in cholesterol-rich, caveolin-containing membrane microdomains . Furthermore, oxytocin receptors are known to initiate different signaling pathways, stimulating or inhibiting cell growth, depending on their location inside or outside caveolar microdomains (43,44). The range of oxytocin concentrations in our saturation binding study (10−10 to 10−7 M) would not detect a second, low affinity binding site in the 10−7 to 10−6 M range as reported by Zhong et al . Since Chen et al have shown that DHA incorporation into membrane phospholipids depletes cholesterol from caveolae , the possibility that DHA in the present study shifts a population of receptors into the low affinity binding state may warrant further investigation. PI turnover and receptor binding sites decreased with 30 µM PUFA treatment, but remained at levels sufficient for oxytocin-stimulated [Ca2+]i mobilization under these conditions.
Prolonged exposure to agonists desensitizes many G protein-coupled receptors (GPCRs), protecting the cell from overstimulation. Following receptor activation, GPCRs are commonly subject to a cycle of internalization and sequestration, resensitization, and recycling back to the plasma membrane , . It is unlikely that the alteration of membrane fatty acid composition affected receptor internalization, however, as we did not detect any differences in the rate of oxytocin receptor internalization upon ligand binding (Figure 5).
In view of the frequently antagonistic actions of dietary AA and DHA, our observation that both n-6 and n-3 LC PUFAs inhibit oxytocin signaling was unexpected. Although we have discovered a generalized LC PUFA effect, translation to human diets and commonly consumed supplements would indicate that this mechanism of action would pertain to fish consumption and fish oil supplementation. AA in the body originates from either synthesis through desaturation and elongation of 18∶2 linoleic acid (LA), an essential fatty acid, or as a result of dietary intake. The intake of LA contributes 5–7% of dietary energy in western nations. A recent review of human literature found that increasing the contribution of LA to as much as 20% of dietary energy does not increase tissue AA in erythrocytes and plasma/serum phospholipids . Meat, fish, and eggs are the major food sources of preformed AA. Studies comparing the fatty acid profile of omnivores and vegetarians have reported only slight differences in the level of AA , .
These data suggest that a moderate variation in intake of AA has no major effect on AA accumulation; thus an effect of AA supplementation observed in cell culture may not be relevant to a physiological diet. In contrast, it is well established that human phospholipid LC n-3 PUFA, such as DHA, responds to dietary intake of DHA and that high linolenic acid intake does not increase phospholipid DHA . It is also important to note that DHA (and also EPA) supplementation displaces AA from human plasma phospholipids in human volunteers consuming a normal Western diet (77).
In conclusion, we have shown that LC PUFAs inhibit oxytocin-stimulated [Ca2+]i mobilization in a cell culture model. These findings, in conjunction with the demonstrated ability of dietary LC PUFAs to alter lipid composition, provide new insight into possible mechanisms pertaining to reports of dietary fish and fish oil consumption prolonging gestation.
The authors wish to acknowledge the assistance of Dr. Chun Ying Ku with the PHM1-41 cell culture.
Conceived and designed the experiments: PYK BMS KGDA. Performed the experiments: PYK MZ YSK. Analyzed the data: PYK MZ YSK BMS KGDA. Contributed reagents/materials/analysis tools: PYK MZ YSK BMS KGDA. Wrote the paper: PYK MZ YSK BMS KGDA.
- 1. Hamilton BE, Martin JA, Ventura SJ (2011) Births: preliminary data for 2010. Natl Vital Stat Rep 60(2):1–36.
- 2. Davidoff MJ, Dias T, Damus K, Russell R, Bettegowda VR, et al. (2006) Changes in the gestational age distribution among U.S. singleton births: impact on rates of late preterm birth, 1992 to 2002. Semin Perinatol 30(1):8–15.
- 3. McCormick MC (1985) The contribution of low birth weight to infant mortality and childhood morbidity. N Engl J Med 312(2):82–90.
- 4. Kramer MS, Demissie K, Yang H, Platt RW, Sauve R, et al. (2000) The contribution of mild and moderate preterm birth to infant mortality. Fetal and Infant Health Study Group of the Canadian Perinatal Surveillance System. Jama 284(7):843–849.
- 5. Allen KG, Harris MA (2001) The role of n-3 fatty acids in gestation and parturition. Exp Biol Med (Maywood) 226(6):498–506.
- 6. Olsen SF, Hansen HS, Sorensen TI, Jensen B, Secher NJ, et al. (1986) Intake of marine fat, rich in (n-3)-polyunsaturated fatty acids, may increase birthweight by prolonging gestation. Lancet 2(8503):367–369.
- 7. Olsen SF, Hansen HS, Sommer S, Jensen B, Sorensen TI, et al. (1991) Gestational age in relation to marine n-3 fatty acids in maternal erythrocytes: a study of women in the Faroe Islands and Denmark. Am J Obstet Gynecol 164(5 Pt 1):1203–1209.
- 8. Olsen SF, Sorensen JD, Secher NJ, Hedegaard M, Henriksen TB, et al. (1992) Randomised controlled trial of effect of fish-oil supplementation on pregnancy duration. Lancet 339(8800):1003–1007.
- 9. Olsen SF, Secher NJ, Tabor A, Weber T, Walker JJ, et al. (2000) Randomised clinical trials of fish oil supplementation in high risk pregnancies. Fish Oil Trials In Pregnancy (FOTIP) Team. Bjog 107(3):382–395.
- 10. Olsen SF, Osterdal ML, Salvig JD, Weber T, Tabor A, et al. (2007) Duration of pregnancy in relation to fish oil supplementation and habitual fish intake: a randomised clinical trial with fish oil. Eur J Clin Nutr 61(8):976–985.
- 11. Olsen SF, Secher NJ (2002) Low consumption of seafood in early pregnancy as a risk factor for preterm delivery: prospective cohort study. Bmj 324(7335):447.
- 12. Smuts CM, Huang M, Mundy D, Plasse T, Major S, et al. (2003) A randomized trial of docosahexaenoic acid supplementation during the third trimester of pregnancy. Obstet Gynecol 101(3):469–479.
- 13. Makrides M, Gibson RA, McPhee AJ, Yelland L, Quinlivan J, et al. (2010) Effect of DHA supplementation during pregnancy on maternal depression and neurodevelopment of young children: a randomized controlled trial. JAMA 304(15):1675–1683.
- 14. Hauner H, Much D, Vollhardt C, Brunner S, Schmid D, et al. (2012) Effect of reducing the n-6:n-3 long-chain PUFA ratio during pregnancy and lactation on infant adipose tissue growth within the first year of life: an open-label randomized controlled trial. Am J Clin Nutr 95(2):383–394.
- 15. Salvig JD, Lamont RF (2011) Evidence regarding an effect of marine n-3 fatty acids on preterm birth: a systematic review and meta-analysis. Acta Obstet Gynecol Scand 90(8):825–838.
- 16. Olson DM, Mijovic JE, Sadowsky DW (1995) Control of human parturition. Semin Perinatol 19(1):52–63.
- 17. Paneth N, Pinto-Martin J (1990) The epidemiology of germinal matrix/intraventricular hemorrhage. In: Reproductive and perinatal epidemiology. Kiely M, editor. Boca Raton: CRC Press. 371–99.
- 18. Committee on Understanding Premature Birth and Assuring Healthy Outcomes (2006) Preterm Birth: Causes, Consequences, and Prevention. Behrman RE, Butler AS, editors. Washington DC: Nat Acad Press. xvi, 772.
- 19. McLaren J, Taylor DJ, Bell SC (2000) Prostaglandin E(2)-dependent production of latent matrix metalloproteinase-9 in cultures of human fetal membranes. Mol Hum Reprod 6(11):1033–1040.
- 20. Ulug U, Goldman S, Ben-Shlomo I, Shalev E (2001) Matrix metalloproteinase (MMP)-2 and MMP-9 and their inhibitor, TIMP-1, in human term decidua and fetal membranes: the effect of prostaglandin F(2alpha) and indomethacin. Mol Hum Reprod 7(12):1187–1193.
- 21. Vadillo-Ortega F, Hernandez A, Gonzalez-Avila G, Bermejo L, Iwata K, et al. (1996) Increased matrix metalloproteinase activity and reduced tissue inhibitor of metalloproteinases-1 levels in amniotic fluids from pregnancies complicated by premature rupture of membranes. Am J Obstet Gynecol 174(4):1371–1376.
- 22. Ulmsten U (1979) Aspects on ripening of the cervix and induction of labor by intracervical application of PGE2 in viscous gel. Acta Obstet Gynecol Scand Suppl 84: 5–9.
- 23. O'Brien WF, Knuppel RA, Cohen GR (1986) Plasma prostaglandin metabolite levels after use of prostaglandin E2 gel for cervical ripening. Am J Obstet Gynecol 155(5):1037–1040.
- 24. Bygdeman M, Kwon S, Wiqvist N (1967) The effect of prostaglandin E1 on human pregnant myometrium in vivo. In: Prostaglandins. Proceedings of the 2nd Nobel Symposium, 1966 June, Stockholm. Bergström S, Samuelsson B, editors. Stockholm: Almqvist and Wiksell. 93–96.
- 25. Karim SM, Hillier K, Somers K, Trussell RR (1971) The effects of prostaglandins E 2 and F 2 alpha administered by different routes on uterine activity and the cardiovascular system in pregnant and non-pregnant women. J Obstet Gynaecol Br Commonw 78(2):172–179.
- 26. Reece MS, McGregor JA, Allen KG, Harris MA (1997) Maternal and perinatal long-chain fatty acids: possible roles in preterm birth. Am J Obstet Gynecol 176(4):907–914.
- 27. Corey EJ, Shih C, Cashman JR (1983) Docosahexaenoic acid is a strong inhibitor of prostaglandin but not leukotriene biosynthesis. Proc Natl Acad Sci U S A 80(12):3581–3584.
- 28. Huang MC, Craig-Schmidt MC (1996) Arachidonate and docosahexaenoate added to infant formula influence fatty acid composition and subsequent eicosanoid production in neonatal pigs. J Nutr 126(9):2199–2208.
- 29. Perez MA, Hansen RA, Harris MA, Allen KG (2006) Dietary docosahexaenoic acid alters pregnant rat reproductive tissue prostaglandin and matrix metalloproteinase production. J Nutr Biochem 17(7):446–453.
- 30. Goldenberg RL, Hauth JC, Andrews WW (2000) Intrauterine infection and preterm delivery. N Engl J Med 342(20):1500–1507.
- 31. Romero R, Emamian M, Wan M, Quintero R, Hobbins JC, et al. (1987) Prostaglandin concentrations in amniotic fluid of women with intra-amniotic infection and preterm labor. Am J Obstet Gynecol 157(6):1461–1467.
- 32. Reece MS, McGregor JA, Allen KG, Mathias MM, Harris MA (1996) Prostaglandins in selected reproductive tissues in preterm and full-term gestations. Prostaglandins Leukot Essent Fatty Acids 55(5):303–307.
- 33. Sadovsky Y, Nelson DM, Muglia LJ, Gross GA, Harris KC, et al. (2000) Effective diminution of amniotic prostaglandin production by selective inhibitors of cyclooxygenase type 2. Am J Obstet Gynecol 182(2):370–376.
- 34. Leaf A, Xiao YF, Kang JX, Billman GE (2005) Membrane effects of the n-3 fish oil fatty acids, which prevent fatal ventricular arrhythmias. J Membr Biol 206(2):129–139.
- 35. Stulnig TM, Huber J, Leitinger N, Imre EM, Angelisova P, et al. (2001) Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition. J Biol Chem 276(40):37335–37340.
- 36. Fan YY, Ly LH, Barhoumi R, McMurray DN, Chapkin RS (2004) Dietary docosahexaenoic acid suppresses T cell protein kinase C theta lipid raft recruitment and IL-2 production. J Immunol 173(10):6151–6160.
- 37. Bruno MJ, Koeppe RE 2nd, Andersen OS (2007) Docosahexaenoic acid alters bilayer elastic properties. Proc Natl Acad Sci U S A 104(23):9638–9643.
- 38. Stulnig TM, Berger M, Sigmund T, Raederstorff D, Stockinger H, et al. (1998) Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains. J Cell Biol 143(3):637–644.
- 39. Zeyda M, Staffler G, Horejsi V, Waldhausl W, Stulnig TM (2002) LAT displacement from lipid rafts as a molecular mechanism for the inhibition of T cell signaling by polyunsaturated fatty acids. J Biol Chem 277(32):28418–28423.
- 40. Klein U, Gimpl G, Fahrenholz F (1995) Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry 34(42):13784–13793.
- 41. Gimpl G, Burger K, Fahrenholz F (1997) Cholesterol as modulator of receptor function. Biochemistry 36(36):10959–10974.
- 42. Gimpl G, Fahrenholz F (2000) Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane. Eur J Biochem 267(9):2483–2497.
- 43. Guzzi F, Zanchetta D, Cassoni P, Guzzi V, Francolini M, et al. (2002) Localization of the human oxytocin receptor in caveolin-1 enriched domains turns the receptor-mediated inhibition of cell growth into a proliferative response. Oncogene 21(11):1658–1667.
- 44. Rimoldi V, Reversi A, Taverna E, Rosa P, Francolini M, et al. (2003) Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization in caveolin-1 enriched domains. Oncogene 22(38):6054–6060.
- 45. Whittington K, Connors B, King K, Assinder S, Hogarth K, et al. (2007) The effect of oxytocin on cell proliferation in the human prostate is modulated by gonadal steroids: implications for benign prostatic hyperplasia and carcinoma of the prostate. Prostate 67(10):1132–1142.
- 46. Fuchs AR, Fuchs F, Husslein P, Soloff MS, Fernstrom MJ (1982) Oxytocin receptors and human parturition: a dual role for oxytocin in the initiation of labor. Science 215(4538):1396–1398.
- 47. Takahashi K, Diamond F, Bieniarz J, Yen H, Burd L (1980) Uterine contractility and oxytocin sensitivity in preterm, term, and postterm pregnancy. Am J Obstet Gynecol 136(6):774–779.
- 48. Monga M, Ku CY, Dodge K, Sanborn BM (1996) Oxytocin-stimulated responses in a pregnant human immortalized myometrial cell line. Biol Reprod 55(2):427–432.
- 49. Burghardt RC, Barhoumi R, Stickney M, Monga M, Ku CY, et al. (1996) Correlation between connexin43 expression, cell-cell communication, and oxytocin-induced Ca2+ responses in an immortalized human myometrial cell line. Biol Reprod 55: 433–438.
- 50. Ku CY, Babich L, Word RA, Zhong M, Ulloa A, et al. (2006) Expression of transient receptor channel proteins in human fundal myometrium in pregnancy. J Soc Gynecol Investig 13: 217–225.
- 51. Barhoumi R, Awooda I, Mouneimne Y, Safe S, Burghardt RC (2006) Effects of benzo-a-pyrene on oxytocin-induced Ca2+ oscillations in myometrial cells. Toxicol Lett 165: 133–141.
- 52. Ciarmela P, Wiater E, Vale W (2008) Activin-A in myometrium: characterization of the actions on myometrial cells. Endocrinology 149: 2506–2516.
- 53. Zhong M, Parish B, Murtazina DA, Ku CY, Sanborn BM (2007) Amino acids in the COOH-terminal region of the oxytocin receptor third intracellular domain are important for receptor function. Am J Physiol Endocrinol Metab 292(4):E977–984.
- 54. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1):497–509.
- 55. Morrison WR, Smith LM (1964) Preparation of Fatty Acid Methyl Esters and Dimethylacetals from Lipids with Boron Fluoride–Methanol. J Lipid Res 5: 600–608.
- 56. Yang M, Wang W, Zhong M, Philippi A, Lichtarge O, et al. (2002) Lysine 270 in the third intracellular domain of the oxytocin receptor is an important determinant for G alpha(q) coupling specificity. Mol Endocrinol 16(4):814–823.
- 57. Yang M, Gupta A, Shlykov SG, Corrigan R, Tsujimoto S, et al. (2002) Multiple Trp isoforms implicated in capacitative calcium entry are expressed in human pregnant myometrium and myometrial cells. Biol Reprod 67(3):988–994.
- 58. Sanborn BM (2007) Hormonal signaling and signal pathway crosstalk in the control of myometrial calcium dynamics. Semin Cell Dev Biol 18(3):305–314.
- 59. Phaneuf S, Rodriguez Linares B, TambyRaja RL, MacKenzie IZ, Lopez Bernal A (2000) Loss of myometrial oxytocin receptors during oxytocin-induced and oxytocin-augmented labour. J Reprod Fertil 120(1):91–97.
- 60. Hallaq H, Sellmayer A, Smith TW, Leaf A (1990) Protective effect of eicosapentaenoic acid on ouabain toxicity in neonatal rat cardiac myocytes. Proc Natl Acad Sci U S A 87(20):7834–7838.
- 61. Grynberg A, Fournier A, Sergiel JP, Athias P (1995) Effect of docosahexaenoic acid and eicosapentaenoic acid in the phospholipids of rat heart muscle cells on adrenoceptor responsiveness and mechanism. J Mol Cell Cardiol 27(11):2507–2520.
- 62. de Jonge HW, Dekkers DH, Bastiaanse EM, Bezstarosti K, van der Laarse A, et al. (1996) Eicosapentaenoic acid incorporation in membrane phospholipids modulates receptor-mediated phospholipase C and membrane fluidity in rat ventricular myocytes in culture. J Mol Cell Cardiol 28(5):1097–1108.
- 63. Ng Y, Barhoumi R, Tjalkens RB, Fan YY, Kolar S, et al. (2005) The role of docosahexaenoic acid in mediating mitochondrial membrane lipid oxidation and apoptosis in colonocytes. Carcinogenesis 26(11):1914–1921.
- 64. Gordon MJ, Campbell FM, Sattar N, Dutta-Roy AK (1997) Interaction of plasma free fatty acids of pregnant mothers with the membrane fatty acid–binding protein of the human placenta. Prostaglandin Leukot Essent Fatty Acids 57(2):232.
- 65. Al MD, van Houwelingen AC, Hornstra G (2000) Long-chain polyunsaturated fatty acids, pregnancy, and pregnancy outcome. Am J Clin Nutr 71(1 Suppl):285S–291S.
- 66. Santoli D, Zurier RB (1989) Prostaglandin E precursor fatty acids inhibit human IL-2 production by a prostaglandin E-independent mechanism. J Immunol 143(4):1303–1309.
- 67. Calder PC, Bevan SJ, Newsholme EA (1992) The inhibition of T-lymphocyte proliferation by fatty acids is via an eicosanoid-independent mechanism. Immunology 75(1):108–115.
- 68. Melkonian KA, Ostermeyer AG, Chen JZ, Roth MG, Brown DA (1999) Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. Many raft proteins are acylated, while few are prenylated. J Biol Chem 274(6):3910–3917.
- 69. Webb Y, Hermida-Matsumoto L, Resh MD (2000) Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J Biol Chem 275(1):261–270.
- 70. Dunstan JA, Mori TA, Barden A, Beilin LJ, Holt PG, et al. (2004) Effects of n-3 polyunsaturated fatty acid supplementation in pregnancy on maternal and fetal erythrocyte fatty acid composition. Eur J Clin Nutr 58(3):429–437.
- 71. Xiao YF, Kang JX, Morgan JP, Leaf A (1995) Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes. Proc Natl Acad Sci U S A 92(24):11000–11004.
- 72. Zhong M, Navratil AM, Clay C, Sanborn BM (2004) Residues in the hydrophilic face of putative helix 8 of oxytocin receptor are important for receptor function. Biochemistry 43(12):3490–3498.
- 73. Chen W, Jump DB, Esselman WJ, Busik JV (2007) Inhibition of cytokine signaling in human retinal endothelial cells through modification of caveolae/lipid rafts by docosahexaenoic acid. Invest Ophthalmol Vis Sci 48(1):18–26.
- 74. Phaneuf S, Asboth G, Carrasco MP, Linares BR, Kimura T, et al. (1998) Desensitization of oxytocin receptors in human myometrium. Hum Reprod Update 4(5):625–633.
- 75. Conti F, Sertic S, Reversi A, Chini B (2009) Intracellular trafficking of the human oxytocin receptor: evidence of receptor recycling via a Rab4/Rab5 “short cycle”. Am J Physiol Endocrinol Metab 296(3):E532–542.
- 76. Rett BS, Whelan J (2011) Increasing dietary linoleic acid does not increase tissue arachidonic acid content in adults consuming Western-type diets: a systematic review. Nutr Metab (Lond) 8: 36.
- 77. Sanders TA, Ellis FR, Dickerson JW (1978) Studies of vegans: the fatty acid composition of plasma choline phosphoglycerides, erythrocytes, adipose tissue, and breast milk, and some indicators of susceptibility to ischemic heart disease in vegans and omnivore controls. Am J Clin Nutr 31(5):805–813.
- 78. Melchert HU, Limsathayourat N, Mihajlovic H, Eichberg J, Thefeld W, et al. (1987) Fatty acid patterns in triglycerides, diglycerides, free fatty acids, cholesteryl esters and phosphatidylcholine in serum from vegetarians and non-vegetarians. Atherosclerosis 65(1–2):159–166.
- 79. Arterburn LM, Hall EB, Oken H (2006) Distribution, interconversion, and dose response of n-3 fatty acids in humans. Am J Clin Nutr 83(6 Suppl):1467S–1476S.