Effects Of Lead On Acid-Sensing Ion Channels Expression In Nervous System

ObjectiveTo observe the effects of lead administration on mRNA and protein expression of ASIC1a,2a and 2b in hippocampus of baby-rats and rat C6 cell line.Methods1.The effects of lead on the growth and development of baby-rats and its ASIC1a,2a and 2b subunits expression The pregnant Wistar rats were randomly assigned to 3 groups,fed with deionized water and lead contained water(0.2%and 1.0%Pb(Ac)₂) respectively.The lead-exposure period was from the pregnancy day 0 till the pups weaned,then the weaning baby-rats were fed with lead water the same as their mothers.At the postnatal day(PN) 0,record the number of baby-rats in each puteus,their body-weight and length.Baby-rats were killed at PN 8d and 50d respectively.Atomic absorption spectrometry was used to determine lead content of their brain.RT-PCR and Western blot were used to observe the mRNA and protein expression of ASIC1a,2a and 2b in hippocampus of baby-rats respectively.2.The effects of lead on ASIC1a,2a and 2b expression in rat C6 cell line After exposed to Pb(Ac)₂ of different concentrations for 24h,cell viability and injury were tested by MTT and LDH release assay,cell apoptosis was tested by flow cytometry, and ASIC1a,2a and 2b mRNA and protein expression were observed by RT- and Real-time PCR and Western blot assay.Intracellular PKC and CaM expression were also examined with RT-PCR and Western blot assay.Results1.The effects of lead on the growth and development of baby-rats and its ASIC1a,2a and 2b subunits expression(1) No significant differences were observed in the number of pups(P＞0.05),while body-weight and length both dose-dependently decreased.(2) The brain lead contents were time- and dose-dependently increased(P＜0.01).(3) Compared with control group,either at PN 8d or PN 50d,ASIC1a mRNA expression of 1.0%Pb(Ac)₂ group were upregulated (P＜0.01),while ASIC1a protein expression of 0.2%and 1.0%Pb(Ac)₂ group were downregulated(P＜0.05),and ASIC2a and 2b mRNA or protein expression of 0.2%, 1.0%lead treatment were not significantly differed with their corr.control group (P＞0.05).These results were confirmed by three independent trails.2.The effects of lead on rat C6 cells and its intracellular ASIC 1a,2a and 2b expression(1) Lead dose-dependently decreased C6 cell viability and increased LDH release(P＜0.01);cell apoptosis showed a non-significant incremental tendency (P＞0.05).(2) Compared to control group,only 1000μmol/L Pb(Ac)₂ increased ASIC1a mRNA expression(P＜0.01),and 50μmol/L and 1000μmol/L Pb(Ac)₂ both decreased ASIC1a protein expression(P＜0.01),while no significant differences were observed in ASIC2a and 2b mRNA and protein expression(P＞0.05).(3) PKC and CaM mRNA and protein expression was dose-dependently decreased from 5μmol/L Pb(Ac)₂ exposure(P＜0.05,P＜0.01).Conclusion1.By maternal exposure through pregnancy and breast-feeding period,lead can affect their growth and development,accumulate in the brain of baby-rats,and interfere with ASIC1a mRNA and protein expression of hippocampus in different developmental stages,suggesting one of the mechanisms of lead neurotoxicity.2.Lead can not only inhibit the proliferation and viability of rat C6 cells,but also increase their permeability.ASIC1a mRNA and protein expression of C6 cells were up- and down-regulated respectively,which contributes to lead neurotoxicity in CNS.3.The effect of lead on CNS ASICs expression is -1a subunit dependent.4.PKC and/or CaM may mediate the effects of lead on ASIC1a expression.In summary,the consistent results of in vitro and in vivo studies demonstrated that lead can selectively interfere with ASIC1a mRNA and protein expression,which provides new clues for the elucidation,prevention and control of lead neurotoxicity.