The roles of acid sphingomyelinase and lipid rafts in EGF receptor clustering during exposure of human amnion epithelial cells to a 50 Hz magnetic field should be investigated.
Previous studies by the authors found that exposing cells to a 50 Hz magnetic field could induce epidermal growth factor receptor (EGFR) clustering, suggesting that EGFR might be one of the cellular targets of EMF (Sun et al. 2008, Ke et al. 2008). In the present study, associated mechanisms of action and signal transduction should be investigated.
Cells were either exposed to the magnetic field or sham exposed. Some samples were pretreated with nystatin (60 µg/ml), a lipid raft inhibitor, or with imipramine (50 µM), a specific inhibitor of acid sphingomyelinase. Moreover, some samples were treated with C2-ceramide (50 µg/ml), the product of the acid sphingomyelinase, after exposure.
A positive control was conducted.
| Exposure | Parameters |
|---|---|
|
Exposure 1:
50 Hz
Exposure duration:
continuous for 5, 15, 30 or 60 minutes
|
|
| Frequency | 50 Hz |
|---|---|
| Type | |
| Waveform | |
| Exposure duration | continuous for 5, 15, 30 or 60 minutes |
| Exposure source | |
|---|---|
| Chamber | cell dishes in exposure in chambers in incubator |
| Setup | exposure system consisted of two exposure chambers (one for exposure and one for sham exposure), which were put in a CO2 incubator, and a set of control devices outside the incubator; each chamber contained a set of square Helmholtz coils (20 cm x 20 cm) which were double wrapped with two lines of copper wire and was encased by mu-metal; a fan on the metal wall ventilated and maintained air and temperature uniform between chamber and incubator; current was fed into the coils with the same direction in the exposure chamber, whereas opposite direction currents were fed into the coils in the sham exposure chamber; the incubator was aired with 95% humidity air and 5% CO2; the temperature in the chambers was maintained at 37.0°C ± 0.1°C throughout the entire exposure period; the difference in temperature between the chambers did not exceed 0.1°C; cell dishes were put in the center of the coils and the magnetic field was perpendicular to the dishes |
| Sham exposure | A sham exposure was conducted. |
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| magnetic flux density | 0.4 mT | - | measured | - | - |
Exposure to the magnetic field for 15 min significantly increased the clustering of EGF receptors in cells compared to sham exposed cells, as it was expected from previous studies. However, pretreatment with nystatin almost completely inhibited the MF-induced EGFR clustering, indicating that it required intact lipid rafts.
Moreover, exposure to the magnetic field for 15 or 30 minutes significantly increased the enzyme activity of acid sphingomyelase compared to sham exposed cells.
It was shown that exposure to the magnetic field for 15 minutes induced lipid raft and acid sphingomyelase clustering and co-location on the cell membrane. The magnetic field-induced EGFR clustering was inhibited completely by imipramine. When C2-ceramide was added to the medium, the MF-induced EGFR clustering was restored.
The authors conclude that the results indicate that acid sphingomyelase mediates the 50 Hz magnetic field-induced EGFR clustering via ceramide generation on lipid rafts in human amnion epithelial cells.
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