Long chain polyunsaturated fatty acids alter oxytocin signaling and receptor density in cultured pregnant human myometrial smooth muscle cells

Abstract

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 PGE(2) and PGF(2α), 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 [Ca(2+)](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.

Figure 1. Membrane fatty acid composition of PHM1-41 cells treated with 10 µM, 30 µM, 100 µM of OA, AA, DHA.

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.

Figure 2. Representative [Ca²⁺]_i traces recorded from PHM1-41 cells.

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.

Figure 3. The change in [Ca²⁺]_i of PHM1-41 cells in response to 25 nM oxytocin.

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.

Figure 4. Total inositol phosphates (IPs) generated in response to 100 nM oxytocin in PHM1-41 cells.

Data are expressed as a percent increase of total IPs over unstimulated basal. Values are mean ± SEM (n = 4 experiments). Bars not sharing common letters are significantly different between treatment groups, p<0.05.

Figure 5. Internalized [³H]-oxytocin in PHM1-41 cells.

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.