Altered ovarian tissue level of lysophosphatidic acid and mRNA expressions of its metabolic enzymes and receptors in rats received gonadotropin-hyperstimulation.

Lysophosphatidic acid (LPA), a well-studied member of the lysophospholipid family, is known to exert an important bio-effect on oocyte maturation and ovulation in mammals. We attempted to determine how follicle maturation in the rat ovary affects the levels of LPA and its precursor lysophospholipids, as well as mRNA levels of LPA-producing and -degrading enzymes and LPA receptors in rats that received gonadotropin-hyper-stimulation. Tissue levels of lysophospholipids were quantified by LC-MS/MS, and relative mRNA expression levels of LPA-producing and -degrading enzymes, and LPA receptors were measured by RT-PCR. Tissue levels of n-6 polyunsaturated LPAs and LPCs were higher in the ovaries of rats after receiving human chorionic gonadotropin, unlike the distinct profiles of n-3 polyunsaturated LPAs, which had lower levels, and LPCs which had higher levels, after the gonadotropin treatment. The effects of different levels of other polyunsaturated lysophospholipids were variable: decreased levels of lysophosphatidylglycerol, and unaltered levels of lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine. The results indicate that expression of mRNA levels of autotaxin and acylglycerol kinase were reduced and expression of lipid phosphate phosphatase 3 was elevated, whereas expressions of two membrane phosphatidic acid phosphatases (A1α and A1ß) and lipid phosphate phosphatase 1 were essentially unaltered in rat ovary at several stages after ovary hyperstimulation. After the gonadotropin treatment, the expression levels of all LPA receptors except LPA3 were decreased at various times. These results are discussed with respect to the physiological processes of the ovarian environment and development in rats.

Extracellular and intracellular productions of lysophosphatidic acids and cyclic phosphatidic acids by lysophospholipase D from exogenously added lysophosphatidylcholines to cultured NRK52E cells.

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that is a notable biomarker of kidney injury. However, it is not clear how LPA is produced in renal cells. In this study, we explored LPA generation and its enzymatic pathway in a rat kidney-derived cell, NRK52E cells. Culturing of NRK52E cells with acyl lysophosphatidylcholine (acyl LPC), or lyso-platelet activating factor (lysoPAF, alkyl LPC) was resulted in increased extracellular level of choline, co-product with LPA by lysophospholipase D (lysoPLD). Their activities were enhanced by addition of calcium ions to the cell culture medium, but failed to be inhibited by S32826, an autotaxin (ATX)-specific inhibitor. Liquid chromatography-tandem mass spectrometric analysis revealed the small, but significant extracellular production of acyl LPA/cyclic phosphatidic acid (cPA) and alkyl LPA/cPA. The mRNA expression of glycerophosphodiesterase (GDE) 7 with lysoPLD activity was elevated in confluent NRK52E cells cultured over 3 days. GDE7 plasmid-transfection of NRK52E cells augmented both extracellular and intracellular productions of LPAs (acyl and alkyl) as well as extracellular productions of cPAs (acyl and alkyl) from exogenous LPCs (acyl and alkyl). These results suggest that intact NRK52E cells are able to produce choline and LPA/cPA from exogenous LPCs through the enzymatic action of GDE7 that is located on the plasma membranes and intracellular membranes.

Phospholipid analysis of two influenza A virus-infected cell lines differing in their viral replication kinetics.

Fluctuations in phospholipid composition in infected cells during influenza A virus replication were analyzed using two different susceptible host cell lines: H292 cells, exhibiting a rapid cytopathic effect, and A549 cells, exhibiting a retarded cytopathic effect. Microarray analysis demonstrated that A549 cells recognized influenza A virus invasion, expression of pathogen recognition genes was affected, and antiviral genes were activated. On the other hand, H292 cells did not display such an antiviral state, and in these cells, rapid virus amplification and a rapid cytopathic effect were observed. Levels of ceramide, diacylglycerol, and lysolipids were higher in virus-infected cells than in the corresponding mock-infected cells at the later stages of infection. The accumulation of these lipids in IAV-infected cells occurred together with viral replication. The relationship between the characteristic features of ceramide, diacylglycerol, and lysolipid in the plasma membrane, where enveloped viruses are released, and their role in viral envelope formation are discussed. Our results indicate that viral replication disturbs cellular lipid metabolism, with consequences for viral replication kinetics.

Hypoxia increases cellular levels of phosphatidic acid and lysophospholipids in undifferentiated Caco-2 cells.

Cancer cells are known to survive in a hypoxic microenvironment by altering their lipid metabolism as well as their energy metabolism. In this study, Caco-2 cells derived from human colon cancer, were found to have elevated intracellular levels of phosphatidic acid and its lysoform, lysophosphatidic acid (LPA), under hypoxic conditions. Our results suggested that the elevation of LPA in Caco-2 cells was mainly due to the combined increases in cellular levels of lysophosphatidylcholine and lysophosphatidylethanolamine by phospholipase A2 and subsequent hydrolysis to LPA by lysophospholipase D. We detected the Ca2+ -stimulated choline-producing activities toward exogenous lysophosphatidylcholines in whole Caco-2 cell homogenates, indicating their involvement in the LPA production in intact Caco-2 cells.

Lysophosphatidic acid production from lysophosphatidylcholine by lysophospholipase D activity of autotaxin in plasma of women with normal and adverse pregnancies.

To identify biomarker lipids causing preterm delivery, we focused on lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA). The results of liquid chromatography-tandem mass spectrometry revealed that plasma levels of LPCs and LPAs were higher in the first and third (T3) trimesters of human normal and adverse pregnancies than in the second trimester, suggesting the direct metabolic conversion of LPC to LPA by lysophospholipase D (lysoPLD) activity of autotaxin. The elevated LPC and LPA levels in women with preterm deliveries in T3 were higher than in women with term deliveries under normal pregnancy in T3. We measured lysoPLD activity of diluted sera of pregnant women by quantification of choline released from exogenous LPC, and found progressive increases of lysoPLD activities in women with normal and adverse pregnancies. Ratios of lysoPLD activities for linoleoyl LPC to that for palmitoyl LPC were found to be decreased in pregnant women compared to that in non-pregnant women. These results may be due to the altered patterns of endogenous modulators for autotaxin and the profiles of the bound metal ion.

Cellular concentrations of plasmalogen species containing a polyunsaturated fatty acid significantly increase under hypoxia in human colorectal cancer, Caco2 cells.

Plasmalogen localized in the raft of mammalian cell membranes plays a role in the storage of polyunsaturated fatty acid (PUFA), and exists to a higher extent in malignant cells that survive, and even grow in hypoxic conditions. The biosynthesis of plasmalogen in mammalian cells has been reported to depend on aerobic conditions. Using liquid chromatography-tandem mass spectrometry, we found that the intracellular concentration of plasmalogen species containing a PUFA at the sn-2-position did not change for two days from the start of hypoxic culture in human colorectal cancer-derived Caco2 cells. At the third day of hypoxia, Caco2 cells showed the average increase rate of 2.6 times in ethanolamine plasmalogen and 2.9 times in choline plasmalogen depending on the molecular species compared with those in the second day of hypoxia. In normoxic culture, there was little quantitative change in any species of both ethanolamine and choline plasmalogens for three days. The up-regulations of mRNA of Ca2+-independent phospholipase A2ß and cytoplasmic phospholipase A2γ as well as the down-regulation of lysoplasmalogenase observed in hypoxia were suggested to be responsible for the increase of plasmalogen in Caco2 cells under hypoxia.

Altered plasma levels of lysophospholipids in response to adrenalectomy of rats.

The aim of this study was to investigate effects of reduced stress hormone by adrenalectomy on rat plasma levels of lysophosphatidic acid (LPA) and other lysophospholipids. We measured activities of lysophospholipase D (lysoPLD) in plasma and lipid phosphate phosphatase (LPP) in blood by determining choline and inorganic phosphate, respectively. LPA, lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS) and lysophosphatodylglycerol were quantified by LC-MS/MS. In adrenalectomized rats, plasma levels of LPA, LPE, LPS and LPI, but not LPC, were increased. The increased level of LPA were due to decreased LPC level, increases plasma activity of lysoPLD toward LPC and decreased LPP activity toward LPA. Daily injections of deoxycoricosterone into rats selectively reversed increased level of LPS. Our results suggest enzymatic mechanism for increased plasma level of LPA, and indicate that the circulating levels of lysophospholipids including LPA in rats are differently affected by artificial suppression of release of adrenergic hormones.

Involvement of acid ceramidase in the degradation of bioactive N-acylethanolamines.

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [14C]ethanolamine, and revealed that overexpressed AC lowered the levels of 14C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.

Identification of human glycerophosphodiesterase 3 as an ecto phospholipase C that converts the G protein-coupled receptor 55 agonist lysophosphatidylinositol to bioactive monoacylglycerols in cultured mammalian cells.

A family of glycerol-based lysolipid mediators comprises lysophosphatidic acid as a representative phospholipidic member but also a monoacylglycerol as a non-phosphorus-containing member. These critical lysolipid mediators are known to be produced from different lysophospholipids by actions of lysophospholipases C and D in mammals. Some members of the glycerophosphodiesterase (GDE) family have attracted recent attention due to their phospholipid-metabolizing activity. In this study, we found selective depletion of lysophosphatidylinositol among lysophospholipids in the culture medium of COS-7 cells transfected with a vector containing glycerophosphodiester phosphodiesterase 2 (GDPD2, GDE3). Thin-layer chromatography and liquid chromatography-tandem mass spectrometry of lipids extracted from GDE3-transfected COS-7 cells exposed to fluorescent analogs of phosphatidylinositol (PI) revealed that GDE3 acted as an ecto-type lysophospholipase C preferring endogenous lysophosphatidylinositol and PI having a long-chain acyl and a short-chain acyl group rather than endogenous PI and its fluorescent analog having two long chain acyl groups. In MC3T3-E1 cells cultured with an osteogenic or mitogenic medium, mRNA expression of GDE3 was increased by culturing in 10% fetal bovine serum for several days, concomitant with increased activity of ecto-lysophospholipase C, converting arachidonoyl-lysophosphatidylinositol, a physiological agonist of G protein-coupled receptor 55, to arachidonoylglycerol, a physiological agonist of cannabinoid receptors 1 and 2. We suggest that GDE3 acts as an ecto-lysophospholipase C, by switching signaling from lysophosphatidylinositol to that from arachidonoylglycerol in an opposite direction in mouse bone remodeling.

Distinct contributions of two choline-producing enzymatic activities to lysophosphatidic acid production in human amniotic fluid from pregnant women in the second trimester and after parturition.

The purpose of this study was to clarify whether human amniotic fluid (AF) contains a significant level of bioactive lysophosphatidic acid (LPA) and, whether autotaxin (ATX) is involved in the production of LPA, if present. Using LC-MS/MS, we found a higher ratio of levels of LPA and its precursor lysophosphatidylcholine (LPC) in AF collected after parturition than that in AF collected at the middle stage of pregnancy. We detected significant choline-producing enzymatic activity toward an exogenous LPC in AF at the middle stage of pregnancy, about half of which was ascribable to ATX. In AF collected after parturition, the ATX-independent choline-producing activity of glycerophosphcholine phosphodiesterase coupled to lysophospholipase A activity was increased in relative to the lysophospholipase D activity of ATX. These results suggest that the increased LPA/LPC ratio in AF at the term of pregnancy was due to not only a moderate increase in the level of LPC, but also an unknown mechanism involving epithelial cells bathed with AF.

A low level of lysophosphatidic acid in human gingival crevicular fluid from patients with periodontitis due to high soluble lysophospholipase activity: Its potential protective role on alveolar bone loss by periodontitis.

We previously detected a submicromolar concentration of lysophosphatidic acid (LPA) in human saliva. Here, we compare LPA concentrations in human gingival crevicular fluid (GCF) from patients with periodontitis and healthy controls, and examine how the local LPA levels are regulated enzymatically. The concentrations of LPA and its precursor lysophospholipids in GCF was measured by liquid chromatography-tandem mass spectrometry. The LPA-producing and LPA-degrading enzymatic activities were measured by quantifying the liberated choline and free fatty acid, respectively. The concentration of LPA in GCF of periodontitis patients was lower than that of healthy controls, due to higher soluble lysophospholipase activity toward LPA. LPA was found to prevent survival of Sa3, a human gingival epithelium-derived tumor cell line, activate Sa3 through Ca2+ mobilization, and release interleukin 6 from Sa3 in vitro. Furthermore, local injection of LPA into the gingiva attenuated ligature-induced experimental alveolar bone loss induced by oral bacteria inoculation in a rat model of periodontitis in vivo. A high concentration of LPA in human GCF is necessary to maintain normal gingival epithelial integrity and function, protecting the progression of periodontitis.

Addition of high load of lysophosphatidic acid to standard and high-fat chows causes no significant changes of its circulating and peripheral tissue levels but affects body weight and visceral fat mass of mice.

Oral administration of lysophosphatidic acid (LPA), a critical intercellular lipid mediator, exerts wound healing and antiulcer effects on gastrointestinal system. To evaluate effects of food-derived LPA on body homeostasis, we measured LPA levels by liquid chromatography-tandem mass spectrometry in chows, feces, plasma, liver, and visceral fat of mice fed a normal or high-fat chow supplemented with or without LPA-rich soybean phospholipids for 30 days. Reductions in daily body weight gains and visceral fat mass were mainly related to lower chow intake by mice fed the LPA-rich high-fat chow, whereas reduced body weight gains and fat mass were mainly related to decreased intestinal triacylglycerol absorption in mice fed LPA-rich chow. Our results showed no significant increase in plasma, liver, or adipose LPA levels, even if a quite high LPA concentration (2.0%) in chows was ingested daily, suggesting limited effects of food-derived LPA on the lumen side of the digestive tract. © 2018 BioFactors, 44(6):548-557, 2018.

Lysophosphatidic acid in medicinal herbs enhances prostaglandin E2 and protects against indomethacin-induced gastric cell damage in vivo and in vitro.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that induces diverse biological responses. Recently, we found that LPA ameliorates NSAIDs-induced gastric ulcer in mice. Here, we quantified LPA in 21 medicinal herbs used for treatment of gastrointestinal (GI) disorders. We found that half of them contained LPA at relatively high levels (40-240 µg/g) compared to soybean seed powder (4.6 µg/g), which we previously identified as an LPA-rich food. The LPA in peony (Paeonia lactiflora) root powder is highly concentrated in the lipid fraction that ameliorates indomethacin-induced gastric ulcer in mice. Synthetic 18:1 LPA, peony root LPA and peony root lipid enhanced prostaglandin E2 production in a gastric cancer cell line, MKN74 cells that express LPA2 abundantly. These materials also prevented indomethacin-induced cell death and stimulated the proliferation of MKN74 cells. We found that LPA was present in stomach fluids at 2.4 µM, which is an effective LPA concentration for inducing a cellular response in vitro. These results indicated that LPA is one of the active components of medicinal herbs for the treatment of GI disorder and that orally administered LPA-rich herbs may augment the protective actions of endogenous LPA on gastric mucosa.

Three lysophosphatidic acids with a distinct long chain moiety differently affect cell differentiation of human colon epithelial cells to goblet cells.

AIM: The intestinal mucus layer helps maintain intestinal homeostasis. In this study, we investigated the effects of lysophosphatidic acids (LPA) on differentiation of human colon carcinoma cell line, HT-29, to goblet cells with and without sodium butyrate, a known differentiation factor for intestinal cells. MAIN METHODS: Number and average size of cells with goblet-like morphology in five photographs per dish were measured for assessment of differentiation of HT-29 cells to goblet cells as well as their relative portion of surface of to whole surface area of the photograph. KEY FINDINGS: Our results revealed that 18:1 LPA enhanced butyrate-induced differentiation of HT-29 cells. Because increased mRNA expression of LPA5 and decreased mRNA expression of LPA6 were observed in HT-29 cells after treatment with butyrate, we explored the effects of alkyl LPA and 20:4 LPA, which show preferentially higher affinities to LPA5 and LPA6, respectively. As a result, the cell differentiation to goblet cell was increased by alkyl LPA but decreased by 20:4 LPA. Further, alkyl LPA and 18:1 LPA, but not 20:4 LPA, were found to reduce the numbers of cells surviving after incubation in a standard culture medium containing 10% fetal calf serum. SIGNIFICANCE: We suggest that the three LPAs positively and negatively affect the differentiation of HT-29 cells to goblet cells, which may be associated with their reduced survival through the activation of distinct LPA receptor(s).

Roe-derived phospholipid administration enhances lymphatic docosahexaenoic acid-containing phospholipid absorption in unanesthetized rats.

Plasma n-3 fatty acids are important as the supplying pool of n-3 fatty acids to various tissues including the brain, although the relationship between dietary n-3 fatty acids and their molecular species in the plasma are not fully clarified. We investigated the intestinal absorption of docosahexaenoic acid (DHA) derived from fish roe phospholipid (Roe-PL) and compared it with fish oil triacylglycerol and free DHA using unanesthetized lymph-cannulated rats. The DHA absorption from intraduodenally administered three samples were not significantly different, whereas Roe-PL administration resulted in a significantly higher level of DHA in the phospholipid fraction than the other two samples administrations. DHA in Roe-PL at the sn-2 position was less hydrolyzed by pancreatin than by purified phospholipase A2 in vitro and simultaneous administration of free DHA and lysophosphatidylcholine did not produce the same results as the Roe-PL administration. Our results indicate that dietary DHA-containing phospholipid is effective to increase the systemic DHA incorporated into phospholipids via intestinal absorption and biosynthesis.

Peripheral tissue levels and molecular species compositions of N-acyl-phosphatidylethanolamine and its metabolites in mice lacking N-acyl-phosphatidylethanolamine-specific phospholipase D.

N-acylethanolamines (NAEs), a class of lipid mediators, are produced from N-acyl-phosphatidylethanolamine (NAPE) by several pathways, including the direct release by NAPE-specific phospholipase D (NAPE-PLD) or the multistep pathway via sn-glycero-3-phospho-N-acylethanolamine (Gp-NAE). Using liquid chromatography-tandem mass spectrometry, we compared peripheral tissue levels of NAPE, Gp-NAE and NAE in NAPE-PLD-deficient (NAPE-PLD-/-) and wild type (WT) mice. NAPE-PLD was suggested to play a major role in the NAPE degradation in heart, kidney, and liver, but not in jejunum, because the NAPE levels except jejunum were significantly higher in NAPE-PLD-/- mice than in WT mice. The deletion of NAPE-PLD failed to alter the NAE levels of these tissues, suggesting its limited role in the NAE production. The enzyme assays with tissue homogenates confirmed the presence of NAPE-PLD-independent pathways in these peripheral tissues. Gp-NAE species having an acyl moiety with 22 carbons and 6 double bonds was enriched in these peripheral tissues. As for sn-2 acyl species of NAPE, 18:2-acyl-containing NAPE species were predominant over 18:1-containing species in heart, liver, and jejunum. Our results show that both molecular species composition of NAPE, NAE and Gp-NAE and their dependencies on Napepld are different among the peripheral tissues, suggesting that each tissue has distinct metabolic pathways and these NAE-containing lipids play tissue-specific roles.

Distribution of glycosylinositol phosphoceramide-specific phospholipase D activity in plants.

Previously, we detected an unknown sphingophospholipid in cabbage leaves and identified it as phytoceramide-1-phosphate (PC1P). We also found an enzyme activity that produces PC1P by glycosylinositol phosphoceramide (GIPC)-specific hydrolysis in cabbage leaves. To characterize the GIPC-specific phospholipase D (GIPC-PLD) activity, we investigated distributions of GIPC-PLD activity in 25 tissues of 10 plants. In most plants, the GIPC-PLD activity was the highest in roots. Young leaves of cabbage and Welsh onion had higher activities than corresponding aged outer leaves. The GIPC-PLD activities in leaves, stems and roots of mung bean were higher in the sprouting stage than in more mature stages. We also examined the distribution of substrate GIPC and product PC1P and found that GIPC was ubiquitously distributed at 50­280 nmol/g (wet wt) in tissues of plants, whereas PC1P was detectable (3­60 nmol/g wet wt.) only in tissues showing considerable GIPC-PLD activity. These results suggest a possibility that GIPC-PLD activity is involved in plant growth.

Daily Intake of High-Fat Diet with Lysophosphatidic Acid-Rich Soybean Phospholipids Augments Colon Tumorigenesis in Kyoto Apc Delta Rats.

BACKGROUND: Oral administration of lysophosphatidic acid (LPA) was shown to attenuate gastric ulceration in rats and mice but aggravate intestinal tumorigenesis in mice. AIMS: The present study examined whether dietary LPA induces or prevents development of colorectal tumor in rats. METHODS: Kyoto Apc Delta rats fed high-fat diet with or without an LPA-rich soybean phospholipid mixture (LSP, 0.1 or 1%) were treated with azoxymethane and dextran sodium sulfate to induce colorectal tumorigenesis. Rats were killed 15 weeks after azoxymethane treatment, and size, total number, location, and severity of colorectal tumors were assessed. Expression of mRNA of LPA receptors in rat colon tissue was assayed. RESULTS: Rats fed the diet supplemented with 1% LSP had a higher number of tumors 2-4 mm long compared than those with or without 0.1% LSP. The mean distance of tumors >4 mm long from the anus was significantly higher than those of tumors <2 and 2-4 mm long in rats fed 1% LSP-supplemented diet. Supplementation of the diet with 0.1% LSP decreased mRNA expression of LPA5 in colon tumors of rats. CONCLUSIONS: Dietary supplementation of LPA-rich phospholipids dose-dependently augmented colorectal tumorigenesis. Decreased expression of LPA5 in colon tumors may be relevant to augmented tumorigenesis.

Calcium-dependent generation of N-acylethanolamines and lysophosphatidic acids by glycerophosphodiesterase GDE7.

N-Acylethanolamines form a class of lipid mediators and include an endocannabinoid arachidonoylethanolamide (anandamide), analgesic and anti-inflammatory palmitoylethanolamide, and appetite-suppressing oleoylethanolamide. In animal tissues, N-acylethanolamines are synthesized from N-acylated ethanolamine phospholipids directly by N-acylphosphatidylethanolamine-hydrolyzing phospholipase D or through multi-step pathways via N-acylethanolamine lysophospholipids. We previously reported that glycerophosphodiesterase (GDE) 4, a member of the GDE family, has lysophospholipase D (lysoPLD) activity hydrolyzing N-acylethanolamine lysophospholipids to N-acylethanolamines. Recently, GDE7 was shown to have lysoPLD activity toward lysophosphatidylcholine to produce lysophosphatidic acid (LPA). Here, we examined the reactivity of GDE7 with N-acylethanolamine lysophospholipids as well as the requirement of divalent cations for its catalytic activity. When overexpressed in HEK293 cells, recombinant GDE7 proteins of human and mouse showed lysoPLD activity toward N-palmitoyl, N-oleoyl, and N-arachidonoyl-lysophosphatidylethanolamines and N-palmitoyl-lysoplasmenylethanolamine to generate their corresponding N-acylethanolamines and LPAs. However, GDE7 hardly hydrolyzed glycerophospho-N-palmitoylethanolamine. Overexpression of GDE7 in HEK293 cells increased endogenous levels of N-acylethanolamines and LPAs. Interestingly, GDE7 was stimulated by micromolar concentrations of Ca2+ but not by millimolar concentrations of Mg2+, while GDE4 was stimulated by Mg2+ but was insensitive to Ca2+. GDE7 was widely distributed in various tissues of humans and mice with the highest levels in their kidney tissues. These results suggested that GDE7 is a novel Ca2+-dependent lysoPLD, which is involved in the generation of both N-acylethanolamines and LPAs.