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

Abstract

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.

Keywords

apoptosis Ca2+fura-2 MDCK cells melittin renal cells thapsigargin

References

1 Pratt JP Ravnic DJ Huss HT Jiang X Orozco BS Mentzer SJ . Melittin-induced membrane permeability: a nonosmotic mechanism of cell death. In Vitro Cell Dev Biol Anim 2005; 41: 349–355.

2 Han HJ Park SH Lee JH Yoon BC Park KM Mar WC . Involvement of oxidative stress in bee venom-induced inhibition of Na+/glucose cotransporter in renal proximal tubule cells. Clin Exp Pharmacol Physiol 2002; 29: 564–568.

3 Berridge MJ . Cell signalling. A tale of two messengers. Nature 1993; 365: 388–389.

4 Berridge MJ . Unlocking the secrets of cell signaling. Annu Rev Physiol 2005; 67: 1–21.

5 Annunziato L Amoroso S Pannaccione A Cataldi M Pignataro G D'Alessio A . Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions. Toxicol Lett 2003; 139: 125–133.

6 Okamoto T Isoda H Kubota N Takahata K Takahashi T Kishi T . Melittin cardiotoxicity in cultured mouse cardiac myocytes and its correlation with calcium overload. Toxicol Appl Pharmacol 1995; 133: 150–163.

7 Sharma SV . Melittin-induced hyperactivation of phospholipase A2 activity and calcium influx in ras-transformed cells. Oncogene 1993; 8: 939–947.

8 Fletcher JE Tripolitis L Beech J . Bee venom melittin is a potent toxin for reducing the threshold for calcium-induced calcium release in human and equine skeletal muscle. Life Sci 1992; 51: 1731–1738.

9 Mix LL Dinerstein RJ Villereal ML . Mitogens and melittin stimulate an increase in intracellular free calcium concentration in human fibroblasts. Biochem Biophys Res Commun 1984; 119: 69–75.

10.

10 Ruben L Akins CD Haghighat NG Xue L . Calcium influx in Trypanosoma brucei can be induced by amphiphilic peptides and amines. Mol Biochem Parasitol 1996; 81: 191–200.

11.

11 Son DJ Ha SJ Song HS Lim Y Yun YP Lee JW . Melittin inhibits vascular smooth muscle cell proliferation through induction of apoptosis via suppression of nuclear factor-kappaB and Akt activation and enhancement of apoptotic protein expression. J Pharmacol Exp Ther 2006; 317: 627–634.

12.

12 Maher S McClean S . Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro. Biochem Pharmacol 2006; 71: 1289–1298.

13.

13 Weston KM Raison RL . Interaction of melittin with a human lymphoblastoid cell line, HMy2. J Cell Biochem 1998; 68: 164–173.

14.

14 Shaposhnikova VV Egorova MV Kudryavtsev AA Levitman MKh . Korystov, YuN. The effect of melittin on proliferation and death of thymocytes. FEBS Lett 1997; 410: 285–288.

15.

15 Urquhart P Pang S Hooper NM . N-glycans as apical targeting signals in polarized epithelial cells. Biochem Soc Symp 2005; 72: 39–45.

16.

16 Jan CR Ho CM Wu SN Tseng CJ . Bradykinin-evoked Ca2+ mobilization in Madin Darby canine kidney cells. Eur J Pharmacol 1998; 355: 219–233.

17.

17 Jan CR Wu SN Tseng CJ . A further investigation of ATP-induced calcium mobilization in MDCK cells. Chin J Physiol 1999; 42: 33–39.

18.

18 Yeh JH Cheng HH Huang CJ Chung HM Chiu HF Yang YL . Effect of anandamide on cytosolic Ca2+ levels and proliferation in canine renal tubular cells. Basic Clin Pharmacol Toxicol 2006; 98: 416–422.

19.

19 Wang JL Lin KL Chen WC Chou CT Huang CJ Liu CS . Effect of celecoxib on Ca2+ fluxes and proliferation in MDCK renal tubular cells. J Recept Signal Transduct Res 2005; 25: 237–249.

20.

20 Putney JW . A model for receptor-regulated calcium entry. Cell Calcium 1986; 7: 1–12.

21.

21 Grynkiewicz G Poenie M Tsien RY . A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440–3450.

22.

22 Merritt JE Jacob R Hallam TJ . Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J Biol Chem 1989; 264: 1522–1527.

23.

23 Thastrup O Cullen PJ Drobak BK Hanley MR Dawson AP . Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 1990; 87: 2466–2470.

24.

24 Ribardo DA Peterson JW Chopra AK . Phospholipase A2-activating protein–an important regulatory molecule in modulating cyclooxygenase-2 and tumor necrosis factor production during inflammation. Indian J Exp Biol 2002; 40: 129–138.

25.

25 Tsien RY . New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 1980; 19: 2396–2404.

26.

26 Yan SM Finato N Artico D Di Loreto C Cataldi P Bussani R . DNA content, apoptosis and mitosis in transplanted human hearts. Adv Clin Path 1998; 2: 205–219.

27.

27 Mene P . Transient receptor potential channels in the kidney: calcium signaling, transport and beyond. J Nephrol 2006; 19: 21–29.

28.

28 Lang F Paulmichl M . Properties and regulation of ion channels in MDCK cells. Kidney Int 1995; 48: 1200–1205.

29.

29 McFadzean I Gibson A . The developing relationship between receptor-operated and store-operated calcium channels in smooth muscle. Br J Pharmacol 2002; 135: 1–13.

30.

30 Baker KJ East JM Lee AG . Mechanism of inhibition of the Ca2+-ATPase by melittin. Biochemistry 1995; 34: 3596–3604.

31.

31 Shorina EA Mast NV Lopina OD Rubtsov AM . Melittin-induced inhibition and aggregation of Ca-ATPase in skeletal muscle sarcoplasmic reticulum: a comparative study. Biochemistry 1997; 36: 13455–13460.

32.

32 Shorina EA Mast NV Storey KB Lopina OD Rubtsov AM . Characteristics of the interaction of melittin with sarcoplasmic reticulum membranes. Biochemistry (Mosc) 1999; 64: 705–713.