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Mechanism of maitotoxin-stimulated phosphoinositide breakdown in HL-60 cells
F Gusovsky1, J A Bitran, T Yasumoto
1Laboratory of BioOrganic Chemistry, National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland.
Abstract:
The marine toxin maitotoxin (MTX) and the chemotactic peptide fMet-Leu-Phe (fMLP) induce the formation of inositol phosphates in HL-60 cells differentiated with dibutyryl cyclic AMP. The increase in [3H]inositol(1,4,5)-trisphosphate is rapid but transient after fMLP stimulation, whereas MTX-induced increase in [3H]inositol(1,4,5)-trisphosphate occurs at a slower rate and is sustained over time. In both cases increases in [Ca++]i, measured with fura-2, parallel the formation of inositol trisphosphate. MTX-mediated stimulation of inositol phosphate formation is inhibited in the absence of calcium, whereas the response to fMLP is not. The calcium ionophore ionomycin stimulates the formation of inositol phosphates in differentiated HL-60 cells. The magnitude of the response is smaller than that obtained with MTX. Ionomycin also induces a rapid but sustained increase of [Ca++]i. In undifferentiated HL-60 cells, neither fMLP nor ionomycin induce significant inositol phosphate formation, and the increase in [Ca++]i elicited by ionomycin is transient. In contrast, the effects of MTX on phosphoinositide breakdown and on [Ca++]i in undifferentiated cells are nearly identical to those elicited by MTX in differentiated cells. In the presence of the intracellular calcium chelator BAPTA, fMLP, ionomycin and MTX still stimulate the generation of inositol phosphates. Guanyl nucleotides and calcium stimulate phospholipase C activity in membrane preparations from differentiated HL-60 cells. fMLP stimulates the enzyme only in the presence of GTP. MTX has no effect on membrane phospholipase C activity.
Insights
Maitotoxin (MTX) and fMet-Leu-Phe (fMLP) trigger distinct inositol phosphate formation in HL-60 cells. MTX causes sustained calcium increases, while fMLP
Area of Science:
- Cellular signaling and calcium homeostasis
- Biochemistry of marine toxins and peptides
- Phosphoinositide metabolism
Background:
- HL-60 cells are a human promyelocytic leukemia cell line commonly used in research.
- Differentiated HL-60 cells respond to various stimuli, including chemotactic peptides and toxins.
- Inositol phosphates and intracellular calcium ([Ca++]i) are critical second messengers in cellular signaling pathways.
Purpose of the Study:
- To investigate and compare the effects of maitotoxin (MTX) and fMet-Leu-Phe (fMLP) on inositol phosphate formation in HL-60 cells.
- To elucidate the role of extracellular and intracellular calcium in MTX- and fMLP-induced signaling.
- To examine the impact of cell differentiation on the cellular responses to these stimuli.
Main Methods:
- Stimulation of differentiated and undifferentiated HL-60 cells with MTX, fMLP, and ionomycin.
- Measurement of [3H]inositol(1,4,5)-trisphosphate formation using radiolabeling techniques.
- Quantification of intracellular calcium ([Ca++]i) changes using the fura-2 fluorescent indicator.
- Assay of phospholipase C activity in membrane preparations.
Main Results:
- Both MTX and fMLP induced inositol trisphosphate formation and [Ca++]i increases in differentiated HL-60 cells.
- MTX elicited a slower, sustained increase in inositol trisphosphate and [Ca++]i, dependent on extracellular calcium.
- fMLP caused a rapid, transient inositol trisphosphate response, independent of extracellular calcium.
- MTX's effects were similar in undifferentiated cells, whereas fMLP's effects were diminished.
- Ionomycin stimulated inositol phosphate formation and sustained [Ca++]i increase in differentiated cells, but transiently in undifferentiated cells.
- MTX did not directly activate membrane phospholipase C, unlike fMLP in the presence of GTP.
Conclusions:
- MTX and fMLP activate distinct signaling pathways leading to inositol phosphate production in HL-60 cells.
- Calcium influx plays a crucial role in MTX-mediated signaling but not in fMLP-mediated signaling.
- Cell differentiation influences the responsiveness of HL-60 cells to fMLP and ionomycin but not MTX.