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

Abstract

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.

Keywords: Choline; Cyclic phosphatidic acid; Glycerophosphodiesterase 7; Lysophosphatidic acid; Lysophosphatidylcholine; NRK52E cells.