Treatment time influences the effects of a low-frequency pulsed electric field on synthesis of tyrosine hydroxylase and dopamine in PC12 cells

摘要

BACKGROUND:Electromagnetic radiation can influence dopamine(DA) synthesis in brain tissues or cells,but electromagnetic frequencies,intensities,and radiation time can produce different effects.In addition,the signal pathway by which electromagnetic radiation influences DA synthesis remains controversial.OBJECTIVE:To determine tyrosine hydroxylase(TH) expression in PC12 cells and DA levels in cell culture media after different periods of low-frequency pulsed electric field(LF-PEF) stimulation,and to determine how LF-PEF signaling stimulates TH synthesis using inhibitors.DESIGN,TIME AND SETTING:A parallel,controlled,cell experiment was performed at the Laboratory of Cell Biology,School of Life Science,East China Normal University,between January and October 2006.MATERIALS:PC12 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences,China.Nerve growth factor was purchased from PeproTech,USA.The protein kinase A inhibitor,H-89,and mitogen-activated protein kinase kinase inhibitor,U0126,were purchased from Sigma,USA.METHODS:(1) Following routine culture in Dulbecco's modified eagle medium,primary PC12 cells were stimulated under LF-PEF(pulse frequency 50 Hz,pulse width 20 μs,peak field strength 1 V/m) for 5,10,15,20,and 30 minutes.(2) Inhibitors(H-89 or U0126,1 μmol/L) were added 30 minutes before LF-PEF stimulation for 10 minutes.MAIN OUTCOME MEASURES:(1) TH expression was determined by Western blot in PC12 cells at 0.5,1,2,3,and 4 days after LF-PEF stimulation.Similarly,DA was measured by high-performance liquid chromatography in media at 2,3,4,or 5 days after LF-PEF.(2) TH expression was detected 1 day after H-89 or U0126 treatment and LF-PEF.RESULTS:(1) Short-term LF-PEF stimulation(5 and 10 minutes) increased TH expression and media DA levels after short-term culture(2 days)(P<0.01),but both parameters decreased with longer culture(3-4 days)(P<0.01).Long-term LF-PEF stimulation(15,20,or 30 minutes) decreased TH and DA synthesis,followed by a rapid increase(P<0.01).(2) H89 could completely inhibit TH expression in PC12 cells stimulated by LF-PEF for 10 minutes,while the inhibition rate of U0126 was 53.2%.CONCLUSION:Short-term LF-PEF first promotes then inhibits,while long-term LF-PEF first inhibits then promotes,TH and DA synthesis.LF-PEF stimulation regulates TH expression primarily by activating protein kinase A to regulate DA synthesis.