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.

Extracellular metabolism-dependent uptake of lysolipids through cultured monolayer of differentiated Caco-2 cells.

Glycerophospholipids are known to be hydrolyzed in the intestinal lumen into free fatty acids and lysophospholipids that are then absorbed by the intestinal epithelial cells. A monolayer of enterocyte-differentiated Caco-2 cell is often used to assess the intestinal bioavailability of nutrients. In this study, we examined how differentiated Caco-2 cells process lysoglycerolipids such as lysophosphatidylcholine (LPC). Our findings were twofold. (1) Caco-2 cells secreted both a lysophospholipase A-like enzyme and a glycerophosphocholine-phosphodiesterase enzyme into the apical, but not basolateral, lumen, suggesting that food-derived LPC is converted to a free fatty acid, sn-glycerol-3-phosphate, and choline through two sequential enzymatic reactions in humans. The release of the latter enzyme was differentiation-dependent. (2) Fatty acid-releasing activities toward exogenous fluorescent LPC, lysophosphatidic acid and monoacylglycerol were shown to be higher on the apical membranes of Caco-2 cells than on the basolateral membranes. These results suggest that human intestinal epithelial cells metabolize lysoglycerolipids by two distinct mechanisms involving secreted or apical-selective expression of metabolic enzymes.

Presence of bioactive lysophosphatidic acid in renal effluent of rats with unilateral ureteral obstruction.

AIMS: Abnormal production of lysophosphatidic acid (LPA), an important lysophospholipid mediator, in the kidney was examined to participate in the pathogenesis of renal fibrosis in rats. The secretory lysophospholipase D activity of autotoxin was considered as a possible pathway for extracellular production of LPA in the pathological renal fluids. MAIN METHODS: In this study of rats with unilateral ureteral obstruction for two weeks, we measured concentrations of LPA and its precursor, lysophosphatidylcholine stored in the urinary bladder and present in the swollen pelvis of the ligated kidney as well as the corresponding blood plasma by liquid chromatography-tandem mass spectrometry. KEY FINDINGS: We found that concentrations of LPA and lysophosphatidylcholine accumulated in the effluent in the swollen pelvis of the ligated kidney of unilateral ureteral obstruction rats were much higher than those in the urinary bladder. The molecular species composition of LPA in the former was considerably different from that in the blood plasma, indicating the involvement of an additional source other than the blood circulation supplying LPA to the effluent in the swollen kidney. A potential mechanism is increased release of LPA from activated renal cells in the ureter-ligated kidney. SIGNIFICANCE: Both pathways for supply of extracellular LPA may participate in the induction and progression of renal tubulofibrosis.