AA-861-induced Ca²⁺ 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 Ca²⁺ mobilization in Madin Darby canine kidney (MDCK) cells has been examined by fluorimetry using fura-2 as a Ca²⁺ indicator. AA-861 at 10-200 μM increased [Ca²⁺](i) concentration dependently. The signal comprised an initial rise and a sustained phase. Ca²⁺ removal inhibited the Ca²⁺ signals by reducing both the initial rise and the sustained phase. In Ca²⁺-free medium, pretreatment with 50 μM AA-861 abolished the Ca²⁺ release induced by thapsigargin (1 μM), an endoplasmic reticulum Ca²⁺ pump inhibitor, and carbonylcyanide m- chlorophenylhydrazone (CCCP; 2 μM), a mitochondrial uncoupler. Pretreatment with CCCP, thapsigargin and gly-phe-β-naphthylamide to deplete the Ca²⁺ stores in mitochondria, the endoplasmic reticulum, and lysosomes, respectively, only partly inhibited AA-861-induced Ca²⁺ release. This suggests AA-861 released Ca²⁺ from multiple internal pools. Addition of 3 mM Ca²⁺ induced a [Ca²⁺](i) rise after pretreatment with 50 μM AA-861 in Ca²⁺-free medium. AA-861 (50 μM)induced internal Ca²⁺ release was not altered by inhibition of phospholipase C with U73122 (2 μM) but was inhibited by 40% by inhibition of phospholipase A₂ with aristolochic acid (40 μM). Collectively, we found that AA-861 increased [Ca²⁺](i) in MDCK cells by releasing Ca²⁺ from multiple internal stores in a manner independent of the formation of inositol-1,4,5-trisphosphate, followed by Ca²⁺ entry from external medium.