Abstract
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 carbonylcyanidem-chlorophenylhydrazone, a mitochondrial uncoupler. Pretreatment with carbonylcyanidem-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.
Footnotes
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Send reprint requests to: C. R. Jan, Ph.D., Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, 386 Ta Chung 1st Rd., Kaohsiung, Taiwan 813. E-mail:crjan{at}isca.vghks.gov.tw
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↵1 This work was supported by grants from National Science Council (NSC89-2320-B-075B-009), VTY Joint Research Program, Tsou's Foundation (VTY88-P3-24), and Veterans General Hospital-Kaohsiung (VGHKS89-13) to C.-R.J.
- Abbreviations:
- [Ca2+]i
- intracellular Ca2+ concentration
- IP3
- inositol-1,4,5-trisphosphate
- MDCK
- Madin-Darby canine kidney
- CCCP
- carbonylcyanide m-chlorophenylhydrazone
- Received September 23, 1999.
- Accepted December 2, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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