Ca2+ mobilization evoked by chloroform in Madin-Darby canine kidney cells

The effect of chloroform on Ca2+ mobilization in Madin-Darby canine kidney cells was examined by using Fura-2 as a Ca2+ probe. Chloroform (24-248 mM) concentration dependently increased intracellular Ca2+ concentration ([Ca2](i)). Ca2+ removal inhibited the Ca2+ signals evoked by 93 to 248 mM chlor oform by reducing both the initial rise and the sustained phase. In Ca2+-fr ee medium, pretreatment with 93 mM chloroform abolished the Ca2+ release in duced by 1 mu M thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor, and partially reduced the Ca2+ release induced by 2 mu M carbonylcyanide m -chlorophenylhydrazone, a mitochondrial uncoupler. Pretreatment with carbon ylcyanide m-chlorophenylhydrazone and thapsigargin to deplete the Ca2+ stor es in mitochondria and the endoplasmic reticulum, respectively, only partia lly inhibited chloroform-induced Ca2+ release. This suggests that chlorofor m released Ca2+ from multiple internal pools. The addition of 3 mM Ca2+ inc reased [Ca2+](i) after pretreatment with 93 mM chloroform in Ca2+-free medi um. La3+ (1 mM) partially inhibited the [Ca2+](i) increase induced by 93 mM chloroform. Chloroform (93 mM)-induced Ca2+ release was not altered when t he formation of inositol-1,4,5-trisphosphate was abolished by U73122 (2 mu M), a phospholipase C inhibitor, but was inhibited by 90% by inhibition of phospholipase A(2) with 40 mM aristolochic acid. Collectively, we found tha t 93 mM chloroform increased [Ca2+](i) in Madin-Darby canine kidney cells b y releasing Ca2+ from multiple stores in a manner independent of the format ion of inositol-1,4,5-trisphosphate, followed by Ca2+ entry from external m edium. Other solvents, such as ethanol, methanol, and DMSO, did not affect the resting [Ca2+](i) at a concentration of 248 mM.