Melittin activates endogenous phospholipase D during cytolysis of human monocytic leukemia cells
Introduction
Melittin is a 26-amino acid peptide that is a major component of bee venom, comprising 50% of its dry weight (Haberman, 1972). The direct cytolytic/necrotic action of melittin has been reported in erythrocytes and mast cells (Haberman, 1972), mouse fibroblast cells Shier, 1979, Lo et al., 1997, skeletal muscle cells Fletcher et al., 1996, Ownby et al., 1997, rat thymocytes Duke et al., 1994, Sakamoto et al., 1996, Shaposhnikova et al., 1997 and in a human lymphoblastoid cell line (Weston and Raison, 1998). Melittin's action was thought to involve membrane pore formation Tosteson and Tosteson, 1981, Vogel and Jahnig, 1986, Laine et al., 1988 or membrane perturbation, resulting in the disruption of the membrane Pawlak et al., 1994, Benachir and Lafleur, 1995 or both DeGrado et al., 1982, Matsuzaki et al., 1997. Cell death was correlated with phospholipase A2 (PLA2) activation (Shier, 1979), since melittin was reported to be an activator of PLA2 both endogenously in intact cells, as well as in in vitro assays (reviewed by Fletcher and Jiang, 1993). Bee venom PLA2 contamination in natural, commercially available melittin was reported to be the basis for 75% of the presumed activation of PLA2 activity (Fletcher et al., 1990). Research in our laboratory indicated that synthetic melittin inhibited bee venom PLA2 activity non-competitively and also caused inhibition of secretory PLA2 (sPLA2) from bee venom, snake venom and bovine pancreas. Further, the PLA2 activity in synovial fluid from rheumatoid arthritis (RA) patients and an uncharacterized phospholipase partially purified from human serum (Saini et al., 1997) were similarly inhibited. The enhanced release of arachidonic acid (AA) and inositides in PC12 cells by synthetic melittin (Choi et al., 1992), supported the earlier views of melittin as an endogenous activator of PLA2 along with phospholipase C (PLC). Hence, the present study was performed with human monocytic leukemia cells (U937) to identify major phospholipid hydrolase(s) activated endogenously in the cells, as well as in the medium, by synthetic melittin at various time points during the course of cytolysis.
Section snippets
Reagents
The Synthetic Antigen Laboratory at M.D. Anderson Cancer Center, Houston, TX, prepared the synthetic melittin. Radiolabelled arachidonic acid (AA) was obtained from American Radiolabelled Chemicals, St. Louis, MO. Unlabeled AA, diacylglycerol (DAG), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) were obtained from Sigma, St. Louis, MO. Phosphatidylethanol (PEt) was purchased, from BioMol Research Laboratories, Plymouth, PA. Silica
Results
The optimum concentration of synthetic melittin required to release the maximum amount of []-AA from the U937 cells (5×105/ml) into the media was 1.0 μM (Fig. 1). Further, at most concentrations, maximum release of []-AA metabolites was achieved within 15 min. Therefore, melittin at a concentration of 1 μM was used throughout this study. Examination of the Giemsa-stained cells revealed that following melittin addition, cells formed loose clumps within one min (Fig. 2B), followed by
Discussion
The present study indicated that melittin caused U937 cell lysis at 10–15 min, which was preceded by hypertrophy at 5 min (Fig. 2). Hypertrophy was reported to be due to a change in membrane permeability to ions (Tosteson and Tosteson, 1981). During hypertrophy, there was an increase in the total (cellular+medium) PEt, PA, and PS, which decreased at 10 min though the level was still higher than the 0 h value in the case of PEt. While total AA increased, total DAG decreased.
Following melittin
Conclusion
The present study showed that melittin activated cellular PLD transiently during pore formation prior to or near to the initiation of cytolysis. Activation of PLD could hydrolyze membrane phospholipids leading to pore formation, or possibly activate, yet another unknown signal transduction cascade causing cell lysis. The initial release of various lipids from the melittin-treated cells could also have resulted from the formation of pores in the cell membrane. In effect, the lytic process
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Acknowledgements
The authors thank Mardelle Susman, Department of Microbiology and Immunology, UTMB, Galveston TX, for editing the manuscript. This study was supported by grant #AI 21463 from the National Institute of Allergy and Infectious Diseases.
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