Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells.
The effect of calmidazolium on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca2+]i and caused cell death in HA59T cells. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations > or =1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1.5 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Calmidazolium induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to L-type Ca2+ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), calmidazolium-induced [Ca2+]i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change calmidazolium-induced [Ca2+]i rises. At concentrations between 1 and 15 microM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via protein kinase C-regulated Ca2+ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.
Liao
Wei-Chuan
WC
Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan.
Huang
Chorng-Chih
CC
Cheng
He-Hsiung
HH
Wang
Jue-Long
JL
Lin
Ko-Long
KL
Cheng
Jin-Shiung
JS
Chai
Kuo-Liang
KL
Hsu
Pei-Te
PT
Tsai
Jeng-Yu
JY
Fang
Yi-Chien
YC
Lu
Yih-Chau
YC
Chang
Hong-Tai
HT
Huang
Jong-Khing
JK
Chou
Chiang-Ting
CT
Jan
Chung-Ren
CR
D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D016257:Fura-2 / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000008:administration & dosage / Q000633:toxicity*; D008113:Liver Neoplasms / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D005456:Fluorescent Dyes; D007093:Imidazoles; D013778:Tetrazolium Salts; C031938:calmidazolium; D011493:Protein Kinase C; D002118:Calcium; D016257:Fura-2