Ca²⁺-dependent Recruitment Of Voltage-gated Sodium Channels Underlies Bilirubin-induced Overexcitation And Neurotoxicity

Objective:Illuminate the bilirubin induced modulation of voltage-gated sodium channel and its impact on neuron vitality in noenatal neurons of medial vestibular nucleus?MVN?.Methods:Prepare brain slices from P4-6 SD rats.Drugs were perfused with gravity perfusion system.We recorded the frequency and amplitude of spontaneous spikes before and after bilirubin incubation with on-cell patch clamp technique,and sodium currents on single MVN cells with whole-cell technique.We also semi-quantify the expression of Nav1.1 on cytomembrane by immunofluorescene staining.The effects of different pretreatment on alteration of spike frequency and amplitude induced by bilirubin were examed(Ca²⁺chelator BAPTA-AM,etc),as well as cell death ratio.All data were analyzed with t test or independent t test by SPSS22.0 software.Results:We examed the influence of bilirubin on spontaneous spikes of medial vestibular nucleus from rat with patch clamp technique,results as listed below:1.Bilirubin significantly increases the firing rate?control:214.80±4.09spikes/min,BIL:249.20±5.62 spikes/min,p=0.002?and amplitude?control:Iinward/Ioutward=3.46±0.03,BIL:Iinward/Ioutward=4.30±0.03,P<0.001?of spontaneous spikes in MVN from neonatal P4-6 rats,this effect cannot be reversed after 3 min wash out(241.60±4.79 spikes/min,P=0.008,I_inward/I_outward=4.27±0.04,P<0.001),indicating that bilirubin induced lasting and irreversible effect on medial vestibular nucleus.2.This phenomenon is dominated by voltage-gated sodium channel,and bilirubin could drastically augment sodium current density(241.60±4.79 spikes/min,P=0.008,I_inward/I_outward=4.27±0.04,P<0.001),accelerate the activation process?Dx:control:5.69±0.19,BIL:4.61±0.32,P=0.009?and depolarize the activation voltage?control:-23.99±0.86 mV,BIL:-30.14±0.73 mV,P<0.001?,while exerts no obvious impact on inactivation process(V_0.5:control:-57.68±1.756 mV;BIL:-55.00±0.89 mV,P=0.216;Dx:control:6.12±0.17,BIL:5.94±0.12,P=0.433).3.Pretreatment with BAPTA-AM could preclude the facilitation of spike frequency?BAPTA-AM:123.00±5.12 spikes/min,BAPTA-AM+BIL:123.80±5.99spikes/min,P=0.757?and ratio of amplitude?BAPTA-AM:2.69±0.03,BAPTA-AM+BIL:2.69±0.02,P=0.846?induced by bilirubin.4.Protein synthsis inhibitor cycloheximide could partially block the increase of spike frequency?cycloheximide:180.80±7.07 spikes/min,cycloheximide+BIL:195.40±7.70 spikes/min,P=0.023?and ratio of amplitude?cycloheximide:2.79±0.02,cycloheximide+BIL:2.90±0.02,P<0.001?.Compared to incubation with bilirubin alone,cycloheximide exhibit partially blockage on spike frequency and ratio of amplitude?frequency:8.08%vs 16.02%,ratio of amplitude:3.85%vs.24.42%?5.Exocytosis inhibitor TAT-NSF700 could prevent bilirubin induced enhancement of frequency?TAT-NSF700:194.00±1.97 spikes/min,TAT-NSF700+BIL:194.80±2.91spikes/min,P=0.723?and ratio of amplitude?TAT-NSF700:2.83±0.02 pA,TAT-NSF700+BIL:2.84±0.02 pA,P=0.633?,indicating bilirubin promote intracellular vesicle recycle to enhance currents of voltage-gated sodium channel.6.Endocytosis inhibitor dynasore could not prevent bilirubin induced enhancement of frequency?Dynasore:234.40±4.20 spikes/min,Dynasore+BIL:264.60±7.39 spikes/min,P=0.011?or amplitude?Dynasore:3.73±0.04,Dynasore+BIL:4.41±0.05,P<0.001?,indicating augment of VGSC current induced by bilirubin is not implemented by reducing endocytosis.After confirming the upregulation of VGSC to cytomembrane underlies bilirubin induced elevation of cellular excitability,we further semiquantify the expression of Nav1.1 on cytomembrane with immunofluorescence technique,and assess the cell vitality with Calcein-AM/PI co-staining to explore the relation between overexcitability and apoptosis.Results as listed below:1.Bilirubin could promote the expression of Nav1.1 on cytomembrane?control:39.46±1.61 AU,BIL:53.75±2.75 AU,P<0.001?,and induce drastic cell death of medial vestibular nucleus?Death ratio:control:30.87±1.51%,BIL:69.13±1.51%,P<0.001?.2.BAPTA-AM could preclude bilirubin induced overexpression of Nav1.1 on cytomembrane?BAPTA-AM:50.66±4.63 AU,BAPTA-AM+BIL:50.79±3.35 AU,P=0.987?,and effectively alleviates cell death induced by bilirubin?BAPTA-AM+BIL:32.50±1.49%,P<0.001?.3.TAT-NSF700 could preclude bilirubin induced overexpression of Nav1.1 on cytomembrane?TAT-NSF700:79.21±6.53 AU,TAT-NSF700+BIL:75.16±3.04 AU,P=0.570?,and effectively alleviates cell death induced by bilirubin?TAT-NSF700+BIL:42.51±1.20%,P<0.001?.4.Proper dose?25?M?of lidocaine could effectively block the neural overexcitability of medial vestibular nucleus induced by bilirubin?control:188.80±9.30 spikes/min,Lidocaine+BIL:201.00±8.79 spikes/min,P=0.226?without oversuppressing the vitality of sodium channel,also it could effectively alleviates cell death induced by bilirubin?BIL:69.13±1.51%,Lidocaine+BIL:37.60±1.32%,P<0.001?.5.BAPTA-AM and lidocaine could both effectively attenuate the basal death ratio during brain slice preparation?Death ratio:Ctl:30.87±1.51%,Lidocaine:22.57±1.25%,P=0.012,BAPTA-AM:22.59±1.40%,P=0.018?.Conclusions:Bilirubin could promote Ca²⁺dependent protein synthesis and exocytosis by increasing intracellular Ca²⁺dose and upregulate the expression of cytomembrane Nav1.1,eventually result in neural overexcitation and death.Nav1.1potentially could be an intervention spot for preventing neonatal hyperbilirubinemia.