Department of Otorhinolaryngology, Affiliated Sixth People's Hospital of Shanghai Jiaotong University, 600 Yishan Road, Shanghai 200233, China.
Eur J Pharmacol. 2011 Jun 25;660(2-3):310-7. doi: 10.1016/j.ejphar.2011.03.017. Epub 2011 Mar 29.
Excitotoxicity contributes to bilirubin-induced central nervous system injury; however, the mechanisms involved remain controversial. Previous studies from our lab have demonstrated that in juvenile rats bilirubin facilitates γ-aminobutyric acid (GABA)/glycinergic synaptic transmission through activation of presynaptic protein kinase A (PKA) in isolated neurons of the ventral cochlear nucleus (VCN). However, the descending mechanism and physiological effects of bilirubin-induced potentiation remain unclear. Here, whole-cell recordings show that 3×10(-6) M bilirubin increased the frequency of both spontaneous (sPSCs) and miniature (mPSCs) GABA/glycinergic postsynaptic currents in VCN neurons of postnatal day 12-14 (P12-14) rats. This action was dependent on the concentration and duration of exposure to bilirubin and was only partially suppressed by 10(-5) M bicuculline. The potentiation effect on mPSCs persisted in a Ca2+-free solution, but was fully occluded by pretreatment with 1,2 bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), an intracellular Ca2+ chelator. Following pretreatment of the neurons with BAPTA-AM, forskolin, a PKA activator, had no effect on the frequency or amplitude of mPSCs. This suggests that Ca2+ release from presynaptic stores is part of the descending pathway of PKA activation and is responsible for biliurbin-induced potentiation of cell activity. Using gramicidin-perforated patch recordings, the reversal potential of GABA-evoked currents (EGABA) was also investigated. The GABA response resulted in depolarization of 12 of 20 recorded VCN neurons from P12-14 rats. Therefore, potentiation of depolarizing GABA/glycinergic transmission by bilirubin may underlie bilirubin excitotoxicity, which may play a role in the hearing impairment observed among hyperbilirubinemic neonates.
细胞外谷氨酸盐兴奋性毒性作用参与了胆红素诱导的中枢神经系统损伤，但其具体机制仍存在争议。本实验室前期研究发现，在幼年大鼠的耳蜗腹核（VCN）分离神经元中，胆红素通过激活突触前蛋白激酶 A（PKA），促进γ-氨基丁酸（GABA）/甘氨酸能突触传递。然而，胆红素诱导的突触传递增强的下行机制和生理效应仍不清楚。本研究通过全细胞膜片钳记录发现，浓度为 3×10(-6)M 的胆红素可增加出生后 12-14 天（P12-14）大鼠 VCN 神经元自发性（sPSCs）和微小（mPSCs）GABA/甘氨酸能突触后电流的频率。这种作用依赖于胆红素的浓度和暴露时间，仅部分被 10(-5)M 荷包牡丹碱抑制。在无钙溶液中，mPSCs 的增强作用持续存在，但经 1,2 双（2-氨苯氧基）乙烷-N,N,N',N'-四乙酸乙二胺四乙酸酯乙酰氧甲酯（BAPTA-AM）预处理后完全阻断，BAPTA-AM 是一种细胞内钙螯合剂。用 BAPTA-AM 预处理神经元后，PKA 激活剂forskolin 对 mPSCs 的频率或幅度没有影响。这表明，来自突触前储存的 Ca2+释放是 PKA 激活的下行途径的一部分，是胆红素诱导的细胞活性增强的原因。使用革兰氏菌素穿孔膜片钳记录，还研究了 GABA 诱导电流（EGABA）的反转电位。在 20 个记录的 P12-14 大鼠 VCN 神经元中，12 个神经元的 GABA 反应导致去极化。因此，胆红素对去极化 GABA/甘氨酸能传递的增强作用可能是胆红素兴奋性毒性的基础，这可能在高胆红素血症新生儿中观察到的听力损伤中起作用。
