Abstract
The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of epoxides from arachidonic acid and other unsaturated fatty acids, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, inflammation and pain. While recent findings suggested complementary biological roles for Nterm-phos, its mode of action is not well understood. Herein, we demonstrate that lysophosphatidic acids are excellent substrates for Nterm-phos. We also showed that sEH phosphatase activity represents a significant (20–60%) part of LPA cellular hydrolysis, especially in the cytosol. This possible role of sEH on LPA hydrolysis could explain some of the biology previously associated with the Nterm-phos. These findings also underline possible cellular mechanisms by which both activities of sEH (EH and phosphatase) may have complementary or opposite roles.
Keywords: soluble epoxide hydrolase, phosphatase, lysophosphatidic acid
The EPHX2 gene encodes for the soluble epoxide hydrolase (sEH), a cytosolic ubiquitous enzyme in mammals [1]. The sEH is expressed in many tissues including liver and kidneys, but also vascular endothelium, leukocytes, red blood cells, smooth muscle cells, adipocytes and the proximal tubule [1][2]. The sEH protein is a homodimer with a monomeric unit of 62.5 kda [3], which has two distinct activities in two separate structural domains of each monomer: the C-terminal epoxide hydrolase activity (Cterm-EH; E.C. 3.3.2.10) and the N-terminal phosphatase activity (Nterm-phos; EC 3.1.3.76) [3]. The C-terminal is the site of the epoxy-fatty acid hydrolysis that is responsible for the biology associated with EPHX2 [1],[4]-[6], while a magnesium dependent hydrolysis of phosphate esters was recently associated with the N-terminal domain of sEH [7]. The Cterm-EH hydrolyzes epoxy-eicosatrienoic acids (EETs) and other epoxy-fatty acids [8], which are potent endogenous signaling molecules [1],[4]. Pharmacological inhibition of Cterm-EH by potent selective inhibitors [9], has resulted in anti-inflammatory [4],[5], anti-hypertensive [10], neuroprotective [11], and cardioprotective [4] effects in animal models. Furthermore, Cterm-EH inhibition resulted in beneficial effects in more complex diseases such as diabetes and metabolic syndrome [12],[13].
There are several lines of evidence indicating a biological role for the sEH phosphatase activity (Nterm-phos). The sEH-null mice that lack both Cterm-EH and Nterm-phos activities have lower cholesterol and steroid levels [14]. Furthermore, in recombinant Hep G2 cells, Cterm-EH activity lowered cholesterol synthesis while Nterm-phos activity increased it [15]. Put together, this suggests that sEH regulates cholesterol levels in vivo and in vitro, and that the Nterm-phos is a potential therapeutic target in hypercholesterolemia-related disorders. Similarly, in recombinant endothelial cells, both Cterm-EH and Nterm-phos activities contribute to growth factor expression and cell growth [16]. In mice, it seems that the Nterm-phos may play a role in the development of hypoxia-induced pulmonary hypertension [17]. The phosphatase activity of sEH has been shown recently to play a pivotal role in the regulation of eNOS activity and NO-mediated endothelial cell functions [18]. Human polymorphism studies have shown that the Arg287Gln polymorph of sEH is associated with the onset on coronary artery calcification in African-American subjects [19], and insulin resistance in type 2 diabetic patients [20]. This SNP (G860A) of sEH reduces both Cterm-EH and Nterm-phos activities [21],[22]. Furthermore, people having a Lys55Arg polymorph of sEH, which has reduced Nterm-phos but increase Cterm-EH, have higher risk of coronary heart diseases. This SNP also increases the long-term risk of ischemic stroke in men [23].
The biological role of an enzyme is intimately linked to the natural substrate(s) it transforms and/or to the products made. Nterm-phos activity was first described to hydrolyze dihydroxy lipid phosphates [7]. Unfortunately, following development of a sensitive LC-MS-MS analytical method, this class of lipid phosphate was never identified in biological fluids and tissue extracts. More recently, terpenic pyrophosphates, which are cholesterol precursors, were shown in vitro to be substrates for Nterm-phos [24],[25], supporting the hypothesis that Nterm-phos is a lipid phosphate phosphatase. However, the observation that Nterm-phos activity increased cholesterol synthesis in cell cultures [15] suggests that terpenic pyrophosphates are not hydrolyzed in vivo by Nterm-phos. Furthermore, hydrolysis of such substrates does not explain the effect of Nterm-phos on cell growth [16]. Recently, Nterm-phos was shown to modulate endothelial cell functions by altering the phosphorylation of endothelial nitric oxide synthase (eNOS) [18]. While no direct proof was presented, this observation lets one hypothesize that Nterm-phos is a protein phosphatase. To distinguish among multiple hypotheses, we screened thirty natural phosphate containing chemicals as Nterm-phos substrates, and assess the role of sEH phosphatase activity in the metabolism of those key compounds in tissue extracts.
Materials and methods
Chemicals
Compounds 1–12 were from Sigma Aldrich, 13–15 from Anaspec (San Jose, CA), compounds 16–29 were obtained from Avanti Polar Lipids (Alabaster, AL), and compound 30 was provided by Alfa Aesar (Ward Hill, MA). The attophos substrate was obtained from Promega (Madison, WI). All other chemicals and solvents were obtained from Fisher Scientific (Pittsburgh, PA) or Sigma Aldrich (St Louis, MO), and were of the highest purity available.
Enzyme preparations
Recombinant human sEH was produced in a baculovirus expression system [26], and purified by affinity chromatography [27]. This enzyme preparation was at least 97% pure, based on SDS–PAGE followed by scanning densitometry. The enzyme preparation was kept at −80°C until use. Protein concentration was quantified by using the Pierce BCA assay using Fraction V bovine serum albumin as the calibrating standard.
Screening and inhibition assay
A library of twenty-nine natural phosphate containing compounds was created in black polystyrene 96-well plates. Furthermore, dodecyl-phosphate, a known inhibitor of Nterm-phos [24], was used as positive control. Each compound was dissolved at 0.5mM in a 80:20 mixture of BisTris/HCl buffer (pH 7.0; 25 mM) and DMSO containing 0.02% of Tween 20, and 1mM MgCl2. Six wells (3 for the blank and 3 for the control 100% activity) received 20 μL of the solvent mixture. For each compound, 20 μL of the solution at 0.5 mM were dispensed in three wells. The exact composition of the plate is given in Fig. S1. The screening of this plate was performed using attophos as substrate [24]. Simplistically, 150 μL of a 2.8 nM solution of purified human sEH in BisTris/HCl buffer (pH 7.0; 25 mM) containing 1mM MgCl2 and 0.1 mg/mL of BSA (buffer A) was added to the wells ([E]final = 2.1 nM; [tested compounds]final = 50 μM). To monitor background hydrolysis, the enzyme was replaced by 150 μL of buffer A. After throughout mixing and pre-incubation at room temperature for 0.5, 15, 30, 60 or 90 minutes, the reaction was started by the addition of 30 μL of a 16.7 μM solution of Attophos in buffer A ([S]final = 2.5 μM). After 10 min incubation at room temperature in the dark, 100 μL of 0.1 M of NaOH in water were added to each well. Following strong mixing, the amount of fluorescent alcohol produced was measured (λex 435 nm, λ em 555 nm, λcutoff 515 nm) with a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at room temperature. The results given are average ± standard deviation from three separate plates.
Inhibition potencies (IC50s) for the Nterm-phos activity were determined using Attophos A ([S]final = 25 μM) as substrate [24]. IC50s for the Cterm-EH activity were determined using racemic cyano(2-methoxynaphthalen-6-yl)methyl trans-(3-phenyl-oxyran-2-yl) methyl carbonate ([S]final = 2.5 μM) [28]. For comparison purpose, the assays for both activities were run with the same enzyme concentration ([E]final = 2.0 nM) and in buffer A. The human sEH was incubated with the inhibitors for 5 min in buffer A at 30 °C prior to substrate addition. The activities were measured by following the formation of the fluorescent products for 10 minutes at 30 °C as described [24]. By definition, IC50 is the concentration of inhibitor that reduces enzyme activity by 50%. Reported IC50 values are the average ± SD (n = 3).
Kinetic assay conditions
Kinetic parameters for a series of lisophoshatidic acids and sphingosine phosphates were determined under steady-state conditions using the purified recombinant human sEH. One microliter of substrate solution in water or ethanol ([S]final from 1.0 to 50 μM; 7 to 8 concentrations used for each substrate) was added to 100 μL of the enzyme solution at 2.6 μg/mL ([E] = 40 nM) in Buffer A. The reaction mixtures were incubated at 30 °C for 5 to 30 minutes. The reactions were then quenched by adding 100 μL of a 50:49:1 mixture of acetonitrile, water and acetic acid (a 99:1 mixture of acetonitrile and acetic acid was used for 1-stearoyl-glycero-3-phosphate) containing 200 nM of hexanoyl-ceramide (CER-6) as internal standard. The quantity of alcohol formed was then determined by Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) as described below. The kinetic constants (VM and KM) were calculated by non-linear fitting of the Michaelis-Menten equation to the results obtained using Sigma Plot version 11.0 (Systat Software Inc.; Chicago, IL). Results are means ± SD (n =3).
Quantification of sphingosine, ceramides and mono-acyl glycerols (MAG)
A Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), which allows the quick quantification of the products was developed based on previously described methods for lipds [29]. A complete description and performance of the method is given in supplementary data. In brief, separation was carried out on short RP-column (20×2.1 mm) yielding in a fast analysis time of less than 2 min per sample. MS detection was carried out after positive electrospray ionization in selected reaction monitoring mode. With dynamic ranges (r2 >0.99) from 3 to 10,000 nM for the products (Table S2), the method allowed the quantitatively monitoring of enzyme conversions rates of at least 0.3% of the substrate.
Tissue preparation
Liver and lungs tissues were collected post-mortem from ≈40 grams male Swiss-Webster mice (n =6). All samples were flash-frozen with liquid nitrogen, and stored at −80 °C until used. After thawing, samples were then homogenized in chilled sodium phosphate buffer (100 mM pH 7.4; buffer B) containing 1mM PMSF, EDTA and DTT. The homogenate was centrifuged for 1h at 100,000g at 4 °C, and the supernatants were collected. The pellets were re-suspended in chilled sodium phosphate buffer B and centrifuged for 1h at 100,000g at 4 °C. The second supernatants were discarded, while the resulting pellets were suspended in buffer B. Both supernatants and pellets solutions were aliquot and flash-frozen with liquid nitrogen, and stored at −80 °C until use.
Activity assay
LPA hydrolysis activity in tissue was measured using compound 16 as substrate. One μL of a 5 mM solution of 1-myristoyl-glycerol-3-phosphate 16 in water ([S]final = 50 μM) was added to 100 μL of the purified human sEH ([E]: 10 nM ≈ 0.7 μg/mL) or of tissue extracts diluted in Buffer A. The reaction mixtures were incubated at 30 °C for 5 to 30 minutes. The reactions were then quenched by adding 100 μL of a 50:49:1 mixture of acetonitrile, water and acetic acid containing 200 nM of CER-6 as internal standard. The quantity of alcohol formed was then determined by LC-MS analysis as described above. The Cterm-EH activity in tissues was measured using [3H]-trans-diphenylpropene oxide (t-DPPO) as substrate [30].
Results
Screening of library of natural phosphates
Table 1 provides the results of Nterm-phos inhibition by a series of natural phosphates. We screened at a concentration of phosphate compounds of 50 μM that is less than the critical micelle concentration for most of the compounds tested [31]. After 30 seconds incubation, we observed significant Nterm-phos inhibition by lipid phosphates 1–3, 16–22, 27 and 29, supporting the role of sEH phosphatase activity as a lipid phosphate phosphatase. Looking at effects on the Cterm-EH under the same conditions, we observed significant inhibition only for compounds 21 (20 ± 3%) and 22 (12 ± 2%). While compounds 1–3 were shown to be Nterm-phos substrates [24][25], neither 16–20 nor 27 and 29 were shown to interact with sEH before. When looking at time dependence of Nterm-phos inhibition by these compounds (Fig. S3), except for 19 and 20, the inhibition decreased over time for the other compounds, suggesting that they are substrates of sEH phosphatase. For 19 and 20, we observed increasing inhibition of Nterm-phos with time, suggesting that their hydrolytic products (C18:1 MAG and C20:4 MAG, respectively) could inhibit Nterm-phos. Using attophos as reporting substrate, we observed that the hydrolytic products of 19 and 20 can inhibit Nterm-phos in a dose dependent manner (Fig. S4). Kinetic analysis revealed a non-competitive mechanism with KI of 30 ± 5 and 5 ± 2 μM for C18:1 MAG and C20:4 MAG, respectively. Because the KI value for C18:1 MAG is around its CMC [31], and because we observed that such concentrations of C18:1 MAG also reduce by ≈50% Cterm-EH, the action of this compound on sEH is neither potent nor specific. On the contrary, the action of C20:4 MAG seems more specific because its KI is below its CMC and that such concentrations did not significantly affect Cterm-EH.
Table 1.
Effect of natural phosphates on Nterm-phos.a
| # | Name | Inhibition (%) |
|---|---|---|
| 1 | geranyl-pyro-phosphate | 30 ± 2* |
| 2 | farnesyl pyro-phosphate | 16 ± 3* |
| 3 | geranyl-geranyl-pyro-phosphate | 16 ± 4* |
| 4 | glucose-6-phosphate | 9 ± 2 |
| 5 | adenosine triphosphate | 0 ± 2 |
| 6 | guanosine triphosphate | 0 ± 2 |
| 7 | creatine phosphate | 9 ± 5 |
| 8 | phospho-threonine | 6 ± 2 |
| 9 | phospho-serine | 8 ± 4 |
| 10 | phospho-tyrosine | 9 ± 2 |
| 11 | α-glycerophosphate | 6 ± 4 |
| 12 | β-glycerophosphate | 9 ± 4 |
| 13 | H-Gly-Arg-Pro-Arg-Thr-Ser-phosphoSer-Phe-ala-glu-Gly-OH | 4 ± 8 |
| 14 | Ac-Glu-Leu-Glu-Phe-phosphoTyr-Met-Asp-Tyr-Glu-NH2 | 7 ± 3 |
| 15 | H-Leu-Lys-Arg-Ala-phosphoThr-Leu-Gly-OH | 9 ± 5 |
| 16 | 1-myristoyl-2-hydroxy-3-glycerophosphate | 63 ± 1* |
| 17 | 1-palmityl-2-hydroxy-3-glycerophosphate | 15 ± 3* |
| 18 | 1-stearyl-2-hydroxy-3-glycerophosphate | 14 ± 1* |
| 19 | 1-oleoyl-2-hydroxy-3-glycerophosphate | 24 ± 4* |
| 20 | 1-arachidonoyl-2-hydroxy-3-glycerophosphate | 32 ± 3* |
| 21 | 1,2-dioleoyl-3-glycerophosphate | 21 ± 2* |
| 22 | glycerol 1-oleoyl-2-hydroxy-3-phospho-glycerol | 12 ± 3* |
| 23 | glycerol 1-oleoyl-2-hydroxy-3-phospho-ethanolamine | 4 ± 8 |
| 24 | glycerol 1-oleoyl-2-hydroxy-3-phospho-choline | 4 ± 4 |
| 25 | glycerol 1-oleoyl-2-hydroxy-3-phospho-serine | 11 ± 6 |
| 26 | glycerol 1-oleoyl-2-hydroxy-3-phospho-inositol | −1 ± 2 |
| 27 | shingosine-1-phosphate | 16 ± 1* |
| 28 | N-octyl-ceramine-phosphate | −2 ± 5 |
| 29 | N-acetyl-ceramide-phosphate | 30 ± 7* |
| 30 | 1-dodecyl-phosphate | 99 ± 1 |
Attophos was substrate ([S] = 2.5 μM). [I] = 50 μM.
Significantly different from control, t test P< 0.01.
Kinetic constants
In order to confirm that compounds 16–20, 27 and 29 are substrates for sEH phosphatase, we developed an analytical method for their hydrolytic products. Using this method and recombinant purified human sEH, we determined the kinetic constants (KM and kcat) for these endogenous lipids. Examples of kinetic data obtained are given in Fig. 1. As shown in Table 2, they are all substrates of Nterm-phos. Compared to the previous reported Nterm-phos substrate 2 [24], the lysophosphatidic acids (LPA; 16–20) are all at least an order of magnitude better substrate for Nterm-phos. Interestingly, for 19 and 20 we observed inhibition at high substrate concentration (see Fig. 1 for 19). For 19, a better fitting was obtained for a cooperative model (n =2) than for the simple Michaelis-Menten equation. Such cooperative effects were previously observed with some Nterm-phos inhibitors [24]. Sphingosine-phosphate 27 and the corresponding N-acetyl ceramide phosphate 29 were also substrates, but they were hydrolyzed by Nterm-phos much less efficiently than the LPAs 16–20.
Fig 1.
Determination of the kinetic constants for 1-myristoyl-2-hydroxy-3-glycerophosphate (16) and 1-oleoyl-2-hydroxy-3-glycerophosphate (19) with the human sEH ([E]final ≈ 40 nM) in bis-Tris HCl buffer (25mM, pH 7.0) containing 0.1 mg/mL of lipid free BSA and 1 mM of MgCl2 at 30 °C. The kinetic constants (KM and VM) were calculated by non-linear fitting of the Michaelis equation using the enzyme kinetic module of SigmaPlot version 9.01 (Systat Software Inc., Chicago IL).
Table 2.
Kinetic constants of recombinant purified human sEH for lipid phosphates.
| # | KM (μM) | kcat (10−3.s−1) | kcat/KM (10−3.s−1.μM−1) | n | r2 |
|---|---|---|---|---|---|
| 2a | 10 ± 1 | 14 ± 1 | 1.3 ± 0.2 | 1 | |
| 16 | 5.1 ± 1.6 | 354 ± 10 | 76 ± 21 | 1 | 0.96–0.99 |
| 17 | 23 ± 4 | 167 ± 16 | 7.6 ± 1.8 | 1 | 0.96–0.99 |
| 18 | 4.2 ± 1.2 | 125 ± 18 | 33 ± 7 | 1 | 0.96–0.98 |
| 19* | 6.9 ± 0.2 | 177 ± 1 | 25 ± 1 | 2 | 0.96–0.99 |
| 20* | 13 ± 2 | 250 ± 16 | 20 ± 2 | 1 | 0.96–0.99 |
| 27 | 31 ± 4 | 18 ± 1 | 0.58 ± 0.04 | 1 | 0.95–0.99 |
| 29 | 67 ± 9 | 136 ± 3 | 2.1 ± 0.3 | 1 | 0.98–0.99 |
Results are average ± standard deviation (n = 3).
Results for compound 2 are from reference [24].
At high substrate concentrations, there was inhibition of Nterm-phos activity.
LPA hydrolysis activity in tissues
In tissues, LPAs hydrolysis has mainly been described being attached to membranes [32]. Because sEH is soluble (mostly cytosolic), one could think that, based on published data, its role in LPA hydrolysis is negligible. To measure such activity, LPAs are classically used at very high dose (millimolar range) with Triton X-100 (1–5mM) to form micelles and in absence of Mg2+ [33]. Interestingly, micelle formation was previously shown to inhibit Cterm-EH activity [1]. Using attophos as reporting substrate, we found that concentrations of Triton X-100 above its CMC of 0.5 mM totally inhibit Nterm-phos. Furthermore, Mg2+ is necessary for Nterm-phos activity [7], suggesting that classic assay conditions for LPA hydrolysis resulted in total inhibition of sEH phosphatase activity.
Thus, to test if the sEH phosphatase plays a significant role in LPA hydrolysis in tissue, we measured the hydrolysis of 16 in Swiss-Webster mice tissue in the presence and absence of Triton X-100. As shown in Table 3, in the absence of Triton X-100 and presence of Mg2+, there is significant LPA hydrolysis activity in the soluble fraction; around 60% of the total LPA activity in the liver and 20% in the lungs. In the presence of Triton X100, the soluble activity is reduced by more than 90%, while one third of the membrane LPA activity remained. Using a specific substrate (t-DPPO [30]), we estimated the amount of sEH in the tissue extract (table 3). There is a very good correlation (r > 0.9) between t-DPPO activity and LPA hydrolysis activity in absence of Triton X-100, which disappears (r < 0.2) when the detergent is present. Put together, these results suggest that sEH has a significant if not major role in intracellular LPA hydrolysis.
Table 3.
Effect of Triton X-100 on LPA hydrolysis activity in Swiss-Webster mice liver and lungs (n =6).
| Liver | lung | |||
|---|---|---|---|---|
| soluble fraction | membranes | soluble fraction | membranes | |
| 1-myristoyl-2-hydroxy-3-glycerophosphate hydrolysis | ||||
|
| ||||
| No Triton X-100 | ||||
| Specific activity (nmol.min−1.mg−1) | 0.98 ± 0.20 | 0.61 ± 0.07 | 0.26 ± 0.07 | 1.23 ± 0.17 |
| Total activity (nmol.min−1.gtissue−1) | 71 ± 22 | 48 ± 21 | 10 ± 2 | 44 ± 7 |
|
| ||||
| with 5 mM Triton X-100 | ||||
| Specific activity (nmol.min−1.mg−1) | 0.04 ± 0.02 | 0.19 ± 0.10 | 0.002 ± 0.004 | 0.39 ± 0.08 |
| Total activity (nmol.min−1.gtissue−1) | 3 ± 2 | 13 ± 5 | 0.04 ± 0.11 | 14 ± 3 |
|
| ||||
| t-DPPO hydrolysis | ||||
| Specific activity (nmol.min−1.mg−1) | 34 ± 4 | 16 ± 2 | 2.1 ± 0.8 | 0.18 ± 0.04 |
| Total activity (nmol.min−1.gtissue−1) | 2465 ± 610 | 1200 ± 400 | 78 ± 18 | 6 ± 2 |
To confirm these results, we measured LPA hydrolysis activity in wild-type and sEH-null C57BL6 mice tissues [14]. We found that there is 99% less LPA hydrolysis activity in the soluble fraction of livers from sEH-null mice (0.021 ± 0.007 nmol.min−1.mg−1; n= 6) than in livers of wild type mice (3.10 ± 0.7 nmol.min−1.mg−1; n =6). Furthermore, we also found a 38% reduction in LPA hydrolysis activity in the soluble fraction of lungs from sEH-null mice (2.4 ± 0.5 nmol.min−1.mg−1; n= 6) than in lungs of wild type mice (3.9 ± 0.7 nmol.min−1.mg−1; n =6).
Discussion
Our results support that sEH phosphatase activity prefers lipid phosphates (Table 1). Interestingly, while LPAs (16–20) inhibited significantly Nterm-phos, the corresponding di-phosphoesters (22–25) have no selective effect on this enzyme, confirming that sEH phosphatase activity prefers mono-phosphate esters [24]. The measurement of the kinetic parameters (Table 2) showed that the LPAs are the best natural substrates for Nterm-phos found so far. Interestingly, LPA hydrolysis has been reported solely for membrane bound enzymes: the lipins and lipid phosphate phosphatases [34],[35]. Compared to kinetic constants published for these later enzymes, sEH hydrolyzes LPAs in vitro one order of magnitude faster. Because the environment in tissues differ widely from in vitro conditions, it is important to estimate the role on an enzyme in tissues. LPA hydrolysis activity has been reported largely in cellular membrane fractions [32], where the lipins and lipid phosphate phosphatases are localized [34],[35]. We showed herein that the absence or very low level of cytosolic LPA hydrolysis reported in these studies and others is caused by inappropriate assay conditions (Triton X-100) that resulted in the inactivation of Nterm-phos [33]. We showed herein than in the absence of Triton X-100 there is a significant amount of soluble LPA hydrolysis activity in tissues (20 to 60% of the total activity). Furthermore, sEH represents the majority of this soluble LPA activity.
By binding to various nuclear receptors, LPAs regulate cell survival, apoptosis, motility, shape, differentiation, gene transcription, and malignant transformation [32],[36]. The pharmacological regulation of LPA levels is a novel approach for the treatment of several cancers, but also inflammation and atherosclerosis leading causes of cardiovascular diseases [36]. The action of sEH on LPA could explain the observed effects of Nterm-phos on cell growth [16],[18]. In addition, LPA inhibits pre-adipocyte differentiation, thus limiting adipogenesis, through interaction with PPARγ [37]. This nuclear receptor plays an essential role in regulating lipid and glucose homeostasis. These facts could explain the role of Nterm-phos in lipid synthesis [14],[15]. It also suggests a complementary role of Nterm-phos to Cterm-EH in the development of diabetes and cardiovascular diseases [1],[2],[12]-[14]. Finally, LPA activates TRPV1 resulting in painful conditions [38]. Interestingly, we showed that Cterm-EH inhibition has analgesic properties [6], suggesting that the phosphatase and epoxide hydrolase activities of sEH may have opposite roles in pain biology.
In conclusion, the significant cellular role of sEH on LPA hydrolysis could explain some of the biology previously associated with Nterm-phos. It also underlines possible cellular mechanism in which LPA hydrolysis could be a cellular mechanism explaining how both activities of the sEH will have either complementary (cardiovascular diseases) or antagonistic (pain perception) roles.
Supplementary Material
Highlights.
Lysophosphatidic acids (LPA) are excellent substrates for sEH phosphatase activity.
sEH phosphatase represents a significant part of LPA cellular hydrolysis.
Due to artefactual inhibition, sEH activity on LPA was previously unaccounted for.
LPA hydrolysis explains biology associated previously with sEH phosphatase.
Acknowledgments
This work was partially funded by NIEHS grant ES02710 and NIEHS Superfund Basic Research Program grant P42 ES04699 and by the Schormueller Foundation Grant to NHS. BDH is a George and Judy Marcus Senior Fellow of the American Asthma Foundation.
Abbreviations
- CMC
critical micelle concentration
- LC-ESI-MS
Liquid chromatography electrospray ionization mass spectrometry
- LPA
lysophosphatidic acid
- MAG
mono-acyl-glycerol
- sEH
soluble epoxide hydrolase
- t-DPPO
trans-diphenylpropene oxide
Footnotes
Disclosure statement
The authors have nothing to disclose.
Declarations of interest
The authors declare no conflicts of interest.
Supplementary data. A detailed description of the LC-MS method used for the quantification of ceramides and mono-acyl-glycerols, as well as a description of the library of phosphates substrates are described in supplementary data associated with this article.
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