Arachidonic acid-activated Na+-dependent Mg2+ efflux in rat renal epithelial cells

Arachidonic acid (AA), a metabolite of membrane phospholipids, and its metabolites are increased in Mg2+ deficiency. We examined whether the extracellular Mg2+ concentration affects AA production and whether AA regulates a putative Na+-dependent Mg2+ efflux pathway in renal epithelial NRK-52E cells. We used the cells cultured in 5 mM Mg2+-containing medium for 2 days because they enable us to detect Na+-stimulated Mg2+ efflux that was not observed in normal culture medium. Removal of extracellular Mg2+ increased AA release both in the absence and presence of extracellular Na+. This was inhibited by methyl arachidonyl fluorophosphonate (MAFP, 10 muM), an inhibitor of cytosolic phospholipase A(2) (cPLA(2)) and Ca2+-independent phospholipase A(2) (iPLA(2)), and bromoenol lactone (BEL, 10 muM), an inhibitor of iPLA(2). However, LY-311727 (10 muM), a secretory phospholipase A(2) (sPLA(2)) inhibitor, had no inhibitory effect. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that NRK-52E cells express cPLA(2) and iPLA(2) mRNAs, but not sPLA(2). In the mag-fura 2 fluorescence measurements, extracellular Mg2+ removal caused slight decrease in the intracellular free Mg2+ concentration ([Mg2+](i)) in the Na+-free condition. The addition of Na+ caused a rapid decrease in [Mg2+](i), indicating the presence of a Na+-dependent Mg2+ efflux pathway. The Na+-dependent [Mg2+](i) decrease was suppressed by MAFP and BEL. On the other hand, AA metabolite inhibitors, nordihydroguaiaretic acid (NDGA) (50 muM), indomethacin (10 muM) and 17-octadecynoic acid (ODYA) (10 muM), enhanced the Na+-dependent [Mg2+](i) decrease. Furthermore, the addition of exogenous AA (30 muM) enhanced the Na+-dependent [Mg2+](i) decrease, which was significantly inhibited by imipramine (0.1 mM), a putative Na+/Mg2+-exchanger inhibitor. These results suggest that extracellular Mg2+ removal elevates AA release mediated mainly by iPLA(2) and that AA upregulates the Na+-dependent Mg2+ efflux in NRK-52E cells. (C) 2003 Elsevier B.V. All rights reserved.