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Maitotoxin and P2Z/P2X(7) purinergic receptor stimulation activate a common cytolytic pore
W P Schilling1, T Wasylyna, G R Dubyak
1Rammelkamp Center for Education and Research, Department of Physiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44109, USA. wschilling@metrohealth.org
Abstract:
The effects of maitotoxin (MTX) on plasmalemma permeability are similar to those caused by stimulation of P2Z/P2X(7) ionotropic receptors, suggesting that 1) MTX directly activates P2Z/P2X(7) receptors or 2) MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore. To distinguish between these two possibilities, the effect of MTX was examined in 1) THP-1 monocytic cells before and after treatment with lipopolysaccharide and interferon-gamma, a maneuver known to upregulate P2Z/P2X(7) receptor, 2) wild-type HEK cells and HEK cells stably expressing the P2Z/P2X(7) receptor, and 3) BW5147.3 lymphoma cells, a cell line that expresses functional P2Z/P2X(7) channels that are poorly linked to pore formation. In control THP-1 monocytes, addition of MTX produced a biphasic increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)); the initial increase reflects MTX-induced Ca(2+) influx, whereas the second phase correlates in time with the appearance of large pores and the uptake of ethidium. MTX produced comparable increases in [Ca(2+)](i) and ethidium uptake in THP-1 monocytes overexpressing the P2Z/P2X(7) receptor. In both wild-type HEK and HEK cells stably expressing the P2Z/P2X(7) receptor, MTX-induced increases in [Ca(2+)](i) and ethidium uptake were virtually identical. The response of BW5147.3 cells to concentrations of MTX that produced large increases in [Ca(2+)](i) had no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- and Bz-ATP-induced pores activate with similar kinetics and exhibit similar size exclusion. Last, MTX-induced pore formation, but not channel activation, is greatly attenuated by reducing the temperature to 22 degrees C, a characteristic shared by the P2Z/P2X(7)-induced pore. Together, the results demonstrate that, although MTX activates channels that are distinct from those activated by P2Z/P2X(7) receptor stimulation, the cytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.
Insights
Maitotoxin (MTX) and P2Z/P2X(7) receptor stimulation both induce cell pores, but MTX activates distinct channels. The study shows MTX and P2Z/P2X(7) receptor stimulation activate indistinguishable cytolytic pores.
Area of Science:
- Cell Biology
- Ion Channel Physiology
- Toxicology
Background:
- Maitotoxin (MTX) affects cell membrane permeability similarly to P2Z/P2X(7) receptor activation.
- This suggests MTX may directly activate P2Z/P2X(7) receptors or trigger a common pore formation pathway.
Purpose of the Study:
- To investigate whether MTX directly activates P2Z/P2X(7) receptors or if both activate a shared cytolytic pore.
- To differentiate between direct receptor activation and common pore formation by MTX.
Main Methods:
- Examined MTX effects on THP-1 cells (with/without P2Z/P2X(7) receptor upregulation).
- Tested MTX on wild-type HEK cells and HEK cells overexpressing P2Z/P2X(7) receptors.
- Assessed MTX impact on BW5147.3 lymphoma cells, which have P2Z/P2X(7) channels poorly linked to pore formation.
- Compared MTX- and Bz-ATP-induced pore kinetics, size exclusion, and temperature sensitivity.
Main Results:
- MTX induced biphasic cytosolic Ca(2+) increases and ethidium uptake in THP-1 cells, comparable in cells with upregulated P2Z/P2X(7) receptors.
- MTX-induced Ca(2+) influx and ethidium uptake were similar in wild-type and P2Z/P2X(7)-expressing HEK cells.
- MTX increased Ca(2+) but not ethidium uptake in BW5147.3 cells, indicating distinct channel activation.
- MTX- and Bz-ATP-induced pores exhibited similar kinetics and size exclusion, and MTX-induced pore formation was temperature-sensitive, like P2Z/P2X(7)-induced pores.
Conclusions:
- MTX activates ion channels distinct from those activated by P2Z/P2X(7) receptor stimulation.
- Despite distinct channel activation, MTX and P2Z/P2X(7) receptor stimulation generate indistinguishable cytolytic/oncotic pores.