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Melittin-induced [Ca2+]i increases and subsequent death in canine renal tubular cells

Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

HH Cheng

Section of Allergy, Immunology and Rheumatology, Chi-Mei Medical Center, Tainan, Taiwan

CJ Huang

Department of Psychiatry, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; Department of Psychiatry, Tian-Sheng Memorial Hospital, Ping-Tong, Taiwan

HC Chang

Department of Urology, College of Medicine, National Taiwan University, Taipei, Taiwan

WC Chen

Department of Surgery, Ping Tung Christian Hospital, Ping Tung, Taiwan

IS Chen

Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

SS Hsu

Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

HT Chang

Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

JK Huang

Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

JS Chen

Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

YC Lu

Department of Orthopaedic Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

CR Jan

Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan crjan{at}isca.vghks.gov.tw

The effect of melittin on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and viability is largely unknown. This study examined whether melittin alters Ca²⁺ levels and causes Ca²⁺-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca²⁺]_i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Melittin at concentrations above 0.5 µM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced by 75% by removing extracellular Ca²⁺. The melittin-induced Ca²⁺ influx was also implicated by melittin-caused Mn²⁺ influx. After pretreatment with 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), melittin-induced Ca²⁺ release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca²⁺ release. At concentrations of 0.5–20 µM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 µM melittin was nearly completely reversed by prechelating cytosolic Ca²⁺ with BAPTA. Melittin at 0.5–2 µM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MDCK cells, melittin induced a [Ca²⁺]_i rise by causing Ca²⁺ release from endoplasmic reticulum and Ca²⁺ influx from extracellular space. Furthermore, melittin can cause Ca²⁺-dependent cytotoxicity in a concentration-dependent manner.

Key Words: apoptosis • Ca2+ • fura-2 • MDCK cells • melittin • renal cells • thapsigargin

Human & Experimental Toxicology, Vol. 27, No. 5, 417-424 (2008)
DOI: 10.1177/0960327108094606


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