Melittin-induced [Ca2+]i increases and subsequent death in canine renal tubular cells.
The effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability is largely unknown. This study examined whether melittin alters Ca(2+) levels and causes Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Melittin at concentrations above 0.5 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 75% by removing extracellular Ca(2+). The melittin-induced Ca(2+) influx was also implicated by melittin-caused Mn(2+) influx. After pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), melittin-induced Ca(2+) release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca(2+) release. At concentrations of 0.5-20 microM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 microM melittin was nearly completely reversed by prechelating cytosolic Ca(2+) with BAPTA. Melittin at 0.5-2 microM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MDCK cells, melittin induced a [Ca(2+)](i) rise by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, melittin can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.
Liu
S I
SI
Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
Cheng
H H
HH
Huang
C J
CJ
Chang
H C
HC
Chen
W C
WC
Chen
I S
IS
Hsu
S S
SS
Chang
H T
HT
Huang
J K
JK
Chen
J S
JS
Lu
Y C
YC
Jan
C R
CR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004336:Drug Antagonism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism; D007684:Kidney Tubules / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008345:Manganese / Q000378:metabolism; D008555:Melitten / Q000819:agonists / Q000633:toxicity*; D013778:Tetrazolium Salts / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D002614:Chelating Agents; D013778:Tetrazolium Salts; D008555:Melitten; D008345:Manganese; D004533:Egtazic Acid; D019284:Thapsigargin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D016257:Fura-2