Type 2 lysophosphatidic acid receptor in gastric surface mucous cells: Possible implication of prostaglandin E2 production.

Lysophosphatidic acid (LPA) is a lipid mediator that induces various cell responses via its specific receptors. Recently, we found that orally administered LPA and phosphatidic acid (PA) ameliorate stress- or aspirin-induced stomach injury. However, the mechanisms underlying these effects have not been elucidated yet. In this study, we examined effect of LPA on prostaglandin (PG) E2 production in MKN74 cells, a gastric cell-line expressing type 2 LPA receptor (LPA2). When the cells were treated with LPA, the level of mRNA of COX-2 but not COX-1 was upregulated. The LPA effect was abolished when the cells were pretreated with pertussis toxin (PTX), suggesting the involvement of receptor(s) coupled with Gi. Pretreatment of MKN74 cells with LPA enhanced the PGE2 production triggered by calcium ionophore A23187. Again, PTX abolished the LPA effect. Fluorescent immunohistochemistry using an antibody against LPA2 showed that surface mucous cells (pit cells) in gastric mucosa of mice express LPA2 on the apical side of the plasma membrane. These results suggest that LPA in the diet or its digestion may contribute to the epithelial integrity of stomach mucosa by enhancement of PGE2 production via activation of LPA2.

Orally administered phosphatidic acids and lysophosphatidic acids ameliorate aspirin-induced stomach mucosal injury in mice.

BACKGROUND: Recent investigations revealed that lysophosphatidic acid (LPA), a phospholipid with a growth factor-like activity, plays an important role in the integrity of the gastrointestinal tract epithelium. AIM: This paper attempts to clarify the effect of orally administered phosphatidic acid (PA) and LPA on aspirin-induced gastric lesions in mice. MATERIALS AND METHODS: Phospholipids, a free fatty acid, a diacylglycerol and a triglyceride at 1 mM (5.7 µmol/kg body weight) or 0.1 mM were orally administered to mice 0.5 h before oral administration of aspirin (1.7 mmol/kg). The total length of lesions formed on the stomach wall was measured as a lesion index. Formation of LPA from PA in the mouse stomach was examined by in vitro (in stomach lavage fluid), ex vivo (in an isolated stomach) and in vivo (in the stomach of a living mouse) examinations of phospholipase activity. RESULTS: Palmitic acid, dioleoyl-glycerol, olive oil and lysophosphatidylcholine did not affect the aspirin-induced lesions. In contrast, phosphatidylcholine (1 mM), LPA (1 mM) and PA (0.1, 1 mM) significantly reduced the lesion index. Evidence for formation of LPA from PA in the stomach by gastric phospholipase A2 was obtained by in vitro, ex vivo and in vivo experiments. An LPA-specific receptor, LPA2, was found to be localized on the gastric surface-lining cells of mice. CONCLUSION: Pretreatment with PA-rich diets may prevent nonsteroidal anti-inflammatory drug-induced stomach ulcers.

Phosphatidic acid production in the processing of cabbage leaves.

Lysophosphatidic acid (LPA) is a lipid mediator involved in various physiological responses, including wound healing. Evidence of the antiulcer activity of LPA has been reported, and soybean LPA at a concentration of 10 µM is effective in reducing stress-induced gastric ulcer. Because LPA can be formed from phosphatidic acid (PA) by digestive phospholipase A2, dietary PA can be considered a potential antiulcer phospholipid. In this study, PA production in cut processing of cabbage leaves was examined. The amounts of PA in sliced, minced, and homogenized cabbage leaves were 107 ± 5, 134 ± 19, and 286 ± 29 nmol PA/g (wet weight), respectively, all being significantly higher than the amount of PA found in intact leaves. Mixing mayonnaise with sliced cabbage dramatically increased the PA content (1586 ± 393 nmol/3 g), indicating phospholipase D activity leaked raw cabbage produced PA. These results indicate that fine cutting raw cabbage leaves and mixing them with foods rich in phospholipids resulted in an abundant production of PA.

Quantification of phosphatidic acid in foodstuffs using a thin-layer-chromatography-imaging technique.

Apical application of lysophosphatidic acid (LPA), a growth-factor-like phospholipid, was shown to prevent or restore gastrointestinal (GI) disorders, such as diarrhea and stomach ulcer, in experimental animals. Because LPA is formed from phosphatidic acid (PA) by the activity of digestive phospholipase A(2), PA is a potential component for dietary treatment of such GI disorders. Here, we quantified PA contained in 38 foodstuffs and 3 herbs by a thin-layer-chromatography-imaging technique. Vegetables belonging to Brassicaceae, such as cabbage leaves (700 nmol/g of wet weight) and Japanese radish leaves (570 nmol/g), contained higher amounts of PA than other foodstuffs. Amounts of PA in fruits, cereals, and starchy root vegetables were below 300 nmol/g. Animal foodstuffs contained low amounts of PA (<60 nmol/g). Interestingly, leaves of Mallotus japonicas, a Japanese edible herb used for treatment of stomach ulcer, had the highest PA (1410 nmol/g) among those examined. The data shown here will be useful for the development of dietary treatment for a damaged GI tract.

A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are growth factor-like lipids having a phosphate group. The concentrations of these mediator lipids in blood are considered to be potential biomarkers for early detection of cancer or vascular diseases. Here, we report a method for simultaneous determination of LPA and S1P using Phos-tag, a zinc complex that specifically binds to a phosphate-monoester group. Although both LPA and S1P are hydrophilic compounds, we found that they acquire hydrophobic properties when they form complexes with Phos-tag. Based on this finding, we developed a method for the enrichment of LPA and S1P from biological samples. The first partition in a two-phase solvent system consisting of chloroform/methanol/water (1:1:0.9, v/v/v) is conducted for the removal of lipids. LPA and S1P are specifically extracted as Phos-tag complexes at the second partition by adding Phos-tag. The Phos-tag complexes of LPA and S1P are detectable by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and quantifiable based on the relative intensities of ions using 17:0 LPA and C17 S1P as internal standards. The protocol was validated by analyses of these mediator lipids in calf serum, a rat brain and a lung. The clean-up protocol is rapid, requires neither thin-layer chromatography (TLC) nor liquid chromatography (LC), and is applicable to both blood and solid tissue samples. We believe that our protocol will be useful for a routine analysis of LPA and S1P in many clinical samples.