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 mu M 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 mu M AA-861 abolished the Ca2+ release induced by thapsigargin (1 mu M), an endoplasmic reticulum Ca2+ pump inhibitor, and carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 mu M), a mitochondrial uncoupler. Pretreatment with CCCP, thapsigargin and gly-phe-beta-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 mu M AA-861 in Ca2+-free medium. AA-861 (50 mu M)induced internal Ca2+ release was not altered by inhibition of phospholipase C with U73122 (2 mu M) but was inhibited by 40% by inhibition of phospholipase A, with aristolochic acid (40 ELM) 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, (C) 1999 academic Press.