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 microM), an inhibitor of cytosolic phospholipase A) (cPLA2) and Ca2+-independent phospholipase A2 (iPLA2), and bromoenol lactone (BEL, 10 microM), an inhibitor of iPLA2. However, LY-311727 (10 microM), a secretory phospholipase A2 (sPLA2) inhibitor, had no inhibitory effect. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that NRK-52E cells express cPLA2 and iPLA2 mRNAs, but not sPLA2. 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 microM), indomethacin (10 microM) and 17-octadecynoic acid (ODYA) (10 microM), enhanced the Na+-dependent [Mg2+]i decrease. Furthermore, the addition of exogenous AA (30 microM) 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 iPLA2 and that AA upregulates the Na+-dependent Mg2+ efflux in NRK-52E cells.
Ikari
Akira
A
Department of Environmental Biochemistry and Toxicology, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Shizuoka 422-8526, Japan. ikari@u-shizuoka-ken.ac.jp
Nakajima
Kumiko
K
Suketa
Yasunobu
Y
Harada
Hitoshi
H
Takagi
Kuniaki
K
D000818:Animals; D017920:Antiporters / Q000037:antagonists & inhibitors; D016718:Arachidonic Acid / Q000378:metabolism*; D001693:Biological Transport, Active; D004848:Epithelium / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism; D054467:Phospholipases A2; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000378:metabolism*
D017920:Antiporters; C046250:sodium-magnesium antiporter; D016718:Arachidonic Acid; D012964:Sodium; D010741:Phospholipases A; D054467:Phospholipases A2; D008274:Magnesium