Arachidonic acid-activated Na +-dependent Mg 2+ efflux in rat renal epithelial cells

Abstract

Arachidonic acid (AA), a metabolite of membrane phospholipids, and its metabolites are increased in Mg 2+ deficiency. We examined whether the extracellular Mg 2+ concentration affects AA production and whether AA regulates a putative Na +-dependent Mg 2+ efflux pathway in renal epithelial NRK-52E cells. We used the cells cultured in 5 mM Mg 2+-containing medium for 2 days because they enable us to detect Na +-stimulated Mg 2+ efflux that was not observed in normal culture medium. Removal of extracellular Mg 2+ increased AA release both in the absence and presence of extracellular Na +. This was inhibited by methyl arachidonyl fluorophosphonate (MAFP, 10 μM), an inhibitor of cytosolic phospholipase A 2 (cPLA 2) and Ca 2+-independent phospholipase A 2 (iPLA 2), and bromoenol lactone (BEL, 10 μM), an inhibitor of iPLA 2. However, LY-311727 (10 μM), 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 Mg 2+ removal caused slight decrease in the intracellular free Mg 2+ concentration ([Mg 2+] i) in the Na +-free condition. The addition of Na + caused a rapid decrease in [Mg 2+] i, indicating the presence of a Na +-dependent Mg 2+ efflux pathway. The Na +-dependent [Mg 2+] i decrease was suppressed by MAFP and BEL. On the other hand, AA metabolite inhibitors, nordihydroguaiaretic acid (NDGA) (50 μM), indomethacin (10 μM) and 17-octadecynoic acid (ODYA) (10 μM), enhanced the Na +-dependent [Mg 2+] i decrease. Furthermore, the addition of exogenous AA (30 μM) enhanced the Na +-dependent [Mg 2+] i decrease, which was significantly inhibited by imipramine (0.1 mM), a putative Na +/Mg 2+-exchanger inhibitor. These results suggest that extracellular Mg 2+ removal elevates AA release mediated mainly by iPLA 2 and that AA upregulates the Na +-dependent Mg 2+ efflux in NRK-52E cells.