The {omega}-3 fatty acid eicosapentaenoic acid elicits cAMP generation in colonic epithelial cells via a "store-operated" mechanism

Abstract

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid abundant in fish oil that exerts a wide spectrum of documented beneficial health effects in humans. Because dietary interventions are relatively inexpensive and are widely assumed to be safe, they have broad public appeal. Their endorsement can potentially have a major impact on human health, but hard mechanistic evidence that specifies how these derivatives work at the cellular level is limited. EPA (50 microM) caused a small elevation of cytoplasmic Ca(2+) concentration ([Ca(2+)]) in intact NCM460 human colonic epithelial cells as measured by fura 2 and a profound drop of [Ca(2+)] within the endoplasmic reticulum (ER) of permeabilized cells as monitored by compartmentalized mag-fura 2. Total internal reflection fluorescence microscopy showed that this loss of ER store [Ca(2+)] led to translocation of the ER-resident transmembrane Ca(2+) sensor STIM1. Using sensitive FRET-based sensors for cAMP in single cells, we further found that EPA caused a substantial increase in cellular cAMP concentration, a large fraction of which was dependent on the drop in ER [Ca(2+)], but independent of cytosolic Ca(2+). An additional component of the EPA-induced cAMP signal was sensitive to the phosphodiesterase inhibitor isobutyl methylxanthine. We conclude that EPA slowly releases ER Ca(2+) stores, resulting in the generation of cAMP. The elevated cAMP is apparently independent of classical G protein-coupled receptor activation and is likely the consequence of a newly described "store-operated" cAMP signaling pathway that is mediated by STIM1.

Fig. 1.

Effect of eicosapentaenoic acid (EPA) on cytoplasmic and endoplasmic reticulum (ER) Ca²⁺, and on the translocation of YFP-STIM1 in NCM460 cells. A: change in intracellular Ca²⁺ in response to 50 μM EPA in the absence and presence of extracellular Ca²⁺ compared with signal elicited by carbachol (“carb”; 100 μM) as measured with fura 2. Error bars indicate ± SE. B: the response to 50 μM EPA (shown here in Ca²⁺-free solutions) was highly variable when assessed at the single cell level. C: experiments with the low-affinity Ca²⁺ indicator mag-fura 2 to measure free Ca²⁺ in the ER lumen in permeabilized cells. The mag-fura 2 ratio decreased, indicating reduction of free Ca²⁺ in the lumen of the ER in response to inositol-1,4,5-trisphosphate (InsP₃) and EPA. D: as in C, the mag-fura 2 ratio change following InsP₃ treatment was not further lowered by subsequent addition of EPA. Ionomycin (5 μM) yielded an additional loss of stored Ca²⁺. E: bottom, time course of YFP-STIM1 translocation to the cell surface following treatment with 50 μM EPA and 5 μM ionomycin as measured using total internal reflection fluorescence (TIRF) microscopy. Intensity was measured from the regions encompassing the entire two cells; top, a-d: TIRF images of punctae at indicated time points; e: conventional epifluorescence image of same cells showing cellular outline (scale bar = 15 μm). Data typical of results from n = 7 cells in 5 independent experiments.

Fig. 2.

cAMP measurements with the FRET- and Epac-based sensor and immunoassay in response to exposure of colonic cell lines to EPA. A: NCM460 cells stably transfected with the EpacH30 probe. The increase in ratio reflects increases in intracellular cAMP; responses to 50 μM EPA followed by 100 μM forskolin (FSK) + 1 mM 3-isobutyl-1-methylxanthine (IBMX). B: response to EPA and 50 nM PGE₂, typical of 28 NCM460 cells, 9 experiments. C: CaCo-2 cells transiently transfected with EpacH30; effect of 50 μM EPA, 50 nM PGE₂, and 100 μM forskolin. D: quantitative competitive immunoassay for cAMP measured in NCM460 and Caco-2 cells in the absence of any treatment (“NT”) or after a 10 min treatment with EPA (50 μM) in Ca²⁺-free solutions (“EPA”). Treatment with EPA yielded a significant increase in cAMP. Data are averages ± SE of n = 3 independent experiments with determinations in duplicate for each condition. **P < 0.02. ***P < 0.003.

Fig. 3.

EPA acts via a store-operated cAMP signaling process in NCM460 cells. A: effect of the Ca²⁺ chelator 1,2 bis (2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM, 40 μM) on the response to EPA as measured with EpacH30. After a first control response, cells were incubated for 20 min with BAPTA-AM on the microscope stage and then challenged again with 50 μM EPA. B: the response to 50 μM EPA was significantly reduced following exposure to ionomycin, and, conversely, the response to ionomycin was abolished when cells were first challenged with 50 μM EPA (C). D: response of HeLa cells to exposure to 50 μM EPA. E: effect of EPA was larger in the absence of extracellular Ca²⁺ (average of 15 cells ± SE). B-D: representative of experiments performed using EpacH90 sensor.

Fig. 4.

Effect of the phosphodiesterase (PDE) inhibitor IBMX on the cAMP response to EPA. A: EPA-induced cAMP response as measured using EpacH90 was eliminated in NCM460 cells after exposure to ionomycin and IBMX. B: EpacH30 response to 50 μM EPA in the presence of the PDE inhibitor IBMX (2 mM) and response to IBMX in the presence of EPA in the same NCM460 cell. C: summary of data expressed as line scatter plots for individual cells. a, b, c, and d are the delta values taken as shown in B. Response to IBMX (a to b): 24 cells, 3 experiments; response to EPA (c to d): 11 cells, 2 experiments.