AA-861-induced Ca(2+) mobilization in Madin Darby canine kidney cells

The effect of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1, 4-benzoquinone (AA-861), a 5-lipoxygenase inhibitor, on CA(2+) mobilization in Madin Darby canine kidney (MDCK) cells has been examined by fluorimetry using fura-2 as a CA(2+) indicator. AA-861 at 10-200 microM increased [CA(2+)](i) concentration dependently. The signal comprised an initial rise and a sustained phase. CA(2+) removal inhibited the CA(2+) signals by reducing both the initial rise and the sustained phase. In CA(2+)-free medium, pretreatment with 50 microM AA-861 abolished the CA(2+) release induced by thapsigargin (1 microM), an endoplasmic reticulum CA(2+) pump inhibitor, and carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler. Pretreatment with CCCP, thapsigargin and gly-phe-beta-naphthylamide to deplete the CA(2+) stores in mitochondria, the endoplasmic reticulum, and lysosomes, respectively, only partly inhibited AA-861-induced CA(2+) release. This suggests AA-861 released CA(2+) from multiple internal pools. Addition of 3 mM CA(2+) induced a [CA(2+)](i) rise after pretreatment with 50 microM AA-861 in CA(2+)-free medium. AA-861 (50 microM)-induced internal CA(2+) release was not altered by inhibition of Phospholipase C with U73122 (2 microM) but was inhibited by 40% by inhibition of Phospholipase A(2) with aristolochic acid (40 microM). Collectively, we found that AA-861 increased [CA(2+)](i) in MDCK cells by releasing CA(2+) from multiple internal stores in a manner independent of the formation of inositol-1,4,5-trisphosphate, followed by CA(2+) entry from external medium.