Vol. 292, Issue 3, 995-1001, March 2000

Ca²⁺ Mobilization Evoked by Chloroform in Madin-Darby Canine Kidney Cells¹

Chung-Ren Jan , Lee-Wei Chen and Ming-Wei Lin

Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, Taiwan (C.-R.J.); Department of Surgery, Veterans General Hospital-Kaohsiung, Taiwan (L.-W.C.); and Department of Biology and Institute of Life Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (C.-R.J., M.-W.L.)

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 chloroform by reducing both the initial rise and the sustained phase. In Ca²⁺-free medium, pretreatment with 93 mM chloroform abolished the Ca²⁺ release induced by 1 µM thapsigargin, an endoplasmic reticulum Ca²⁺ pump inhibitor, and partially reduced the Ca²⁺ release induced by 2 µM carbonylcyanide m-chlorophenylhydrazone, a mitochondrial uncoupler. Pretreatment with carbonylcyanide m-chlorophenylhydrazone and thapsigargin to deplete the Ca²⁺ stores in mitochondria and the endoplasmic reticulum, respectively, only partially inhibited chloroform-induced Ca²⁺ release. This suggests that chloroform released Ca²⁺ from multiple internal pools. The addition of 3 mM Ca²⁺ increased [Ca²⁺]_i after pretreatment with 93 mM chloroform in Ca²⁺-free medium. La³⁺ (1 mM) partially inhibited the [Ca²⁺]_i increase induced by 93 mM chloroform. Chloroform (93 mM)-induced Ca²⁺ release was not altered when the formation of inositol-1,4,5-trisphosphate was abolished by U73122 (2 µM), a phospholipase C inhibitor, but was inhibited by 90% by inhibition of phospholipase A₂ with 40 µM aristolochic acid. Collectively, we found that 93 mM chloroform increased [Ca²⁺]_i in Madin-Darby canine kidney cells by releasing Ca²⁺ from multiple stores in a manner independent of the formation of inositol-1,4,5-trisphosphate, followed by Ca²⁺ entry from external medium. Other solvents, such as ethanol, methanol, and DMSO, did not affect the resting [Ca²⁺]_i at a concentration of 248 mM.