A Preliminary Study On The Role Of Acid Sphingomyelinase And Ceramide In Receptor Clustering Induced By 50-Hz Magnetic Fields

An extremely frequency low electromagnetic field(ELF EMF) is usually defined as an alternative electromagnetic field with frequency below 300 Hz.Power frequency magnetic fields(MF) which is generated by 50 or 60 Hz alternating current belongs to ELF EMF,and it usually exists around household electric appliances,industrial and agricultural electric settings,electrical power generation,transmission and distribution lines.In recent decades,with rapid development of electric power and widely use of electric appliances,power frequency electromagnetic fields in people's daily life and working environment is increasing greatly,which is becoming a kind of main potential pollution followed with water,air pollution and solid garbage,and had been set by United Nations Conference on Human Environment as the pollution must be controlled.Numbers of studies have shown that 50 Hz MF,as a physical stimulus,could produce wide biological/deleterious effects,which might be involved in tumor development,cardiovascular diseases,endocrine system diseases,reproductive and developmental anomalies and so on.But it is still unclear now how cells or organisms transform a physical stimuli signal into a biological signal,and then induce final effects, and it is becoming an urgent problem needed to be solved.Several researches showed that cell receptor clustering induced by external stimulus is essential for the activation of membrane receptors and downstream signaling pathway.In the previous studies,we found that exposure ceils to 50 Hz MF could induce clustering of epidermal growth factor receptor(EGFR) and tumor necrosis factor receptor(TNFR) on cellular membrane,and EGFR clustering induced by 50 Hz MF was related closely to lipid rafts which is an detergent-insoluble glycolipid-enriched complex on cellular membrane.So we supposed that lipid rafts,especially membrane receptors,could possibly be one of the target sites in which ELF-MF interacts with cells or organisms.Related studies suggested that the activity of acid sphingomyelinase(A-SMase) and the content of ceramide in cell membrane are importantly involved in triggering receptor's clustering on membrane.The formation of liPid raft signaling platforms and membrane receptors clustering induced by many external stimulus may represent one of important mechanism of transmembrane signaling process.The general procedures possibly include A-SMase activation and translocation into cell membrane,ceramide generation,fusion of lipid rafts,and the receptors clustering on signaling platforms. These external stimulus also include some non-ligand physical factors,such as ultraviolet ray C band(UV-C),ionizing radiationγray,and so on.Ceramide as an important second messenger is a basic composition for sphingomyelin construction, which is basic component of cell membrane.With the development of the study on cell membrane signaling,it showed that sphingolipid,especially ceramide,played an. important role in cell transmembrane signaling.Activated A-SMase can translocate into cell membrane and hydrolyze sphingomyelin into ceramide.Related study found the level of reactive oxygen species(ROS) after external stimulation is essential for A-SMase activation and translocation.We also found 50 Hz MF exposure could induce A-SMase co-location with lipid raft on cell membrane in previous experiment.So,we presumed that A-SMase,ceramide and ROS might play key roles in triggering receptor clustering induced by 50-Hz MF.To investigate further the detail mechanisms of 50-Hz MF-induced receptor clustering,in this study,human amniotic(FL) cells were exposed to a 50 Hz sinusoidal magnetic field at 0.4 mT,the relationship among 50-Hz MF-induced epidermal growth factor receptor(EGFR) clustering,A-SMase and ceramide was explored,and the effects of 50-Hz MF on cellular ROS was measured using active oxygen detection technology in FL cells.The role of A-SMase in EGFR clustering induced by 50 Hz magnetic fieldsIn the present study,immunofluorescence method and confocal microscopy were used to observe EGFR clustering.FL cells were cultured on a glass cover slip placed in a petri dish and divided into three groups randomly,including Imipramine(the inhibitor of A-SMase) pretreatment group,cells were pretreated with 50μmol/L Imipramine for 4 h;DMSO group,cells were pretreated with DMSO for 4 h,and non-pretreatment group. Each group was set up three subgroups for the following treatment respectively:a) sham exposure,b) MF exposure,cells were exposed to 50 Hz,0.4 mT MF for 15 min,and c) positive control,cells were treated with 100 ng/ml EGF for 15 min.After treatment, cells on a glass cover slip were rinsed with 0.1 M ice-cold phosphate buffered saline (PBS),fixed with 4%paraformaldehyde for 15 min,sealed with goat serum for 2 h, incubated with antibodies of EGF receptor for 2 h and goat anti-rabbit IgG-FITC for 1 h. After every step,cells were rinsed mildly with PBS for 3-5 times.Finally,the cover slips attached cells were sealed on glass slide.Clustering of EGF receptors was analyzed with a confocal microscopy(Leica,TCS-SP Heidelburg,Germany).The percentage of cells with EGF receptor clustering was counted and compared. Experiment was repeated at least three times.The results showed that in DMSO pretreatment group and non-pretreatment group,both EGF treatment and 50 Hz MF exposure could induce EGFR clustering.It presented one or severl big bright-green spots or 'cap' shape sopts on the cell surface.On the contrary,EGFR almost displayed a homogenous distribution state throughout the membrane in the sham exposure cells. The percentage of EGFR clustering cells in EGF treatment and 50 Hz MF exposure were significant increase compared to sham exposure.However,Imipramine pretreatment could eliminate the effect induced by EGF treatment or 50 Hz MF exposure.The results showed that DMSO almost did not influence the EGFR clustering. It is suggested clearly that the clustering of EGFR induced by 50 Hz MF depends on A-SMase activity.The role of ceramide in EGFR clustering induced by 50 Hz magnetic fieldsIn the experiment,FL cells were cultured on a glass cover slip placed in a petri dish and pretreated with Imipramine for 4 h.Cells were divided into two groups randomly, including ceramide pretreatment group which cells were prctreated with ceramide for 30 min and non-pretreatment group.Each group was set up three subgroups for the following treatment respectively:a) sham exposure,b) MF exposure,cells were exposed to 50 Hz,0.4 mT MF for 15 min,and c) positive control,cells were treated with 100 ng/ml EGF for 15 min.Following treatment,cells were harvested and prepared as above protocol.Finally,clustering of EGF receptors was analyzed with a confocal microscopy.The percentage of cells with EGF receptor clustering was counted and compared.Results showed that after Imipramine pretreatment for 4 h,both EGF treatment and 50 Hz MF exposure couldn't enhance EGFR clustering,there is no significant statistic difference(P＞0.05) compared to sham exposure.While pretreated with ceramide for 30 min,the percentage of cells with EGFR clustering was significant increase(p＜0.05) caused by either EGF treatment or 50 Hz MF exposure,compared to sham exposure.It is suggested that ceramide,as the hydrolyzate from A-SMase might participate in the process of EGFR clustering induced by 50 Hz ME The effects of 50Hz magnetic fields on intracellular ROS productionIn this experiment,we studied the effects of 50 Hz MF exposure on intracellular ROS production using active oxygen detection technology.FL cells were cultured in black 96-multiwell plates for 24 h,then loaded with fluorescent probe DCFH-DA and cultured 20 min.After this,cells were refreshed culture medium,divided into five groups and exposed to 50 Hz MF at 0.4 mT for 0(sham exposure),5,15,30 min or treated with positive agent(provided in agent Kit) for 30 min respectively.Every group was performed in eight-well cells and set the same wells cells as the background in which no fluorescent probe DCFH-DA was loaded.All groups were gathered at the same time,and the intracellular ROS content was detected at 525 nm wavelength under 488 nm wavelength provocative light by fluorescence microplate reader.The results showed that FL cells exposing to 50 Hz MF for 5,15 or 30 min could enhance the ROS production,the statistic difference is significant(p＜0.05,p＜0.01) comparing to sham exposure group.It is suggested that 50 Hz MF exposure could enhance cellular ROS production in FL cells.ConclusionBased on the present studies,we got the following conclusions:1.EGFR clustering induced by 50 Hz MF depended on A-SMase activity.2.Ceramide,as the hydrolyzate from A-SMase might participate in the process of EGFR clustering induced by 50 Hz ME3.Exposure to 50 Hz MF at 0.4 mT could enhance intracellular ROS level in FL cells.