作者姓名：张 晨   
  论文题目：背根神经节细胞的刺激分泌耦联和细胞膜的动态平衡   
  作者简介：张 晨，男，1976年9月出生，1998年9月师从于中国科学院上海生命科学研究院周专研究员，于2003年8月获博士学位（硕博连读生）。   
     
  中 文 摘 要   
  　

动作电位，对于可兴奋细胞，包括神经元和一些内分泌细胞，是一种非常重要的生理信号。  现在被普遍接受的观点是当可兴奋细胞受到动作电位的刺激后，细胞的电压依赖性钙离子通道被打开，在动作电位的下降相引起细胞外钙离子的内流从而升高细胞内的游离钙离子浓度，触发许多细胞内的过程，譬如说神经递质分泌、信号转导和基因表达等。  其中含有递质的囊泡与细胞膜的融合是受动作电位严格调控的一个重要过程，在突触传递、激素分泌中起到了基本的作用，而动作电位对这个过程的调控被广泛认为是通过内流的钙离子起作用的。 

为了更全面地了解动作电位在细胞分泌中的作用，我们使用膜电容检测、微碳纤电极记录、光学测量以及免疫学的方法，研究了动作电位本身（而不是由它导致的内流Ca2+）在细胞分泌过程中所起的作用以及其对随后的胞饮过程的调控。

1)  在大鼠DRG神经元的胞体，我们的研究表明除了钙离子依赖的细胞分泌以外，还存在着一种钙离子不依赖的、电压依赖的细胞分泌。从传统的观点来看，去极化引起的递质分泌中去极化的贡献只是打开电压依赖性的钙离子通道。 我们现在报道的CIVDS显示去极化本身在细胞的分泌过程中也有作用，它可以引起一个快速的、恢复时程很短的细胞分泌；与钙离子引起的恢复时程较长的分泌相比，可能起到了一种相互补充的作用。 在我们测试过的DRG神经元(mice, SD和Wistar Rat)中，CIVDS都存在。 而在肾上腺嗜铬细胞和一些神经突触中，递质分泌可以被钙离子通道的阻断剂完全阻断，显示并没有CIVDS的存在。 在别的系统中，包括神经突触，是否存在CIVDS是一个非常有趣的问题。

2)  在背根神经节神经元上对于细胞分泌触发的胞饮过程的研究发现，存在一种钙离子不敏感的快速胞饮（RE）过程。 RE的时程在秒级，而且是与CIVDS耦联的，CIVDS引起的额外的细胞膜的增加在10s后可以被RE回收86 ± 3%。 RE与刺激的长度和幅度都没有关系，而只与相应的CIVDS的大小线形相关，这个结果暗示在DRG神经元上RE是由胞吐所触发的，而不是由去极化引起的别的信号所引起的。 尽管单个去极化的参数并不能影响RE，但是一串去极化的频率可以调控胞饮的动力学。 与在神经肌肉接头和毛细胞上报道的低频刺激可以增加胞饮不同，在DRG上高频刺激增加胞饮，而低频刺激会减缓胞饮。 磷酸化不能影响低频刺激引起的相对较慢的胞饮，但是可以减缓高频以及单个去极化引起的胞饮。 这种作用可以通过PKA的实验所模拟，显示PKA途径可能参与了DRG细胞上的频率依赖性的快速胞饮。 抑制细胞内的内源性dynamin的活性并不能影响DRG细胞的快速胞饮，但是可以影响受体介导的内吞。 除了钙离子不依赖的快速胞饮，DRG细胞还存在一种钙离子依赖的慢速胞饮，其时程在分钟量级。 慢速胞饮通常不能被小的胞吐所诱发，通常在细胞大量胞吐后才会发生。 慢速胞饮与相对应的胞吐也是线形相关的，而且可以被胞外钙离子被钡离子替换所抑制。 关于细胞骨架在胞饮中的贡献的报道有很多。 破坏细胞内的微管结构据报道可以阻断肾上腺嗜铬细胞的钙离子依赖的胞饮。 我们的结果也显示用nocodazole破坏DRG细胞的微管结构后，细胞的慢速胞饮就被大大减弱了。 我们的这些结果对于胞饮的调控会起到帮助的作用，尤其是清楚的揭示了胞饮的频率依赖性，以及磷酸化和PKA途径在其中的作用。 这对后续的研究会有重要的影响。  

3)  我们发现在背根神经节神经元激活delta阿片受体可以升高细胞内的钙离子，从而引起神经肽的分泌和DOR的上膜。 激活DOR引起的DOR的上膜是依赖于LDCV与细胞膜的融合和细胞内的钙离子升高。 激活DORs可以引起胞内IP3钙库的释放，和一种不通过电压依赖性钙通道的外钙内流；从而引起DOR的上膜和神经肽的分泌。 上膜的DORs可以继续放大这种作用，形成一个正反馈，从而耗竭细胞内的DORs。 这个发现对于疼痛的治疗可能能起到一定的指导作用。

   综上所述，这些研究结果表明, 动作电位对递质分泌的调控并不是完全通过动作电位引起的钙离子内流起作用的，膜电位的变化本身在背根神经节细胞上对递质分泌存在直接的作用。  这些结果对于进一步的理解动作电位对细胞分泌的直接作用有重要的意义，并且对于“细胞膜的膜电位变化对细胞的囊泡循环过程是否存在直接的调控作用”这个问题起到了很好的启发作用并给出了一些解释。

 关键词：背根神经节细胞、钙离子、细胞分泌、细胞胞饮、磷酸化、PKA、阿片受体

 
  
  Stimulus-Secretion Coupling and Membrane Homeostasis in Dorsal Root Ganglion Neurons
Chen Zhang

ABSTRACT   
          Action potential (AP) is a very important physiological signal in excitable cells including neurons and neuroendocrine cells.  It is widely accepted that AP depolarize the plasma membrane and open voltage-dependent calcium channels (VDCCs), the calcium influx through VDCCs trigger the fusion of vesicles with plasma membrane and subsequent transmitter release.  This fusion event is assumed as a strictly Ca2+-regulated process, and plays important roles in transmitter release, synaptic transmission and et al. 

Combined capacitance measurement, amperometric recording and optical detection of secretion, we want to address the question that if AP play some additional role (s) in the exocytosis process except only opening VDCCs.

The main results obtained are listed below:

1)        In the present study we found that, in addition to the Ca2+ dependent secretion (CDS), membrane depolarization alone can trigger a novel form of Ca2+-independent secretion (CIVDS) in somata of rat DRG neurons.  The conventional view of depolarization-induced vesicular secretion is that the sole contribution of the membrane depolarization to exocytosis is to open voltage-gated Ca2+ channels1.  The novel form of CIVDS found here may serve a complementary role to that of CDS by conferring a fast depolarization-induced response with a rapid recovery in DRG neurons.  CIVDS exists in all DRG neurons we have tested thus far (mouse, SD and Wistar rats).  In contrast, secretion from chromaffin cells and many presynaptic terminals is completely abolished by Ca2+ channel blockers21, suggesting the absence of CIVDS in these cells.  The molecular mechanism underlying CIVDS is unknown.  It is possible that the docked secretory vesicles are associated with some intrinsic plasma membrane proteins that sense the transmembrane potential.  Depolarization may cause a conformational change of these proteins, which facilitates exocytosis of the docked vesicles in a Ca2+-independent manner.  Future studies should address the kinetic differences between CDS and CIVDS, relative contributions of CIVDS and CDS under physiological conditions, potential differences in the vesicle types associated with CIVDS and CDS, and the molecular mechanisms underlying CIVDS in DRG neurons.  Furthermore, it would be of interest to determine whether CIVDS also exists in other systems.

2)        We found following CIVDS rapid endocytosis occurred to retrieve the added membrane in seconds.  The Ca2+-independent rapid endocytosis was strict-coupled with CIVDS.  The added membrane was retrieved by 86 ± 3% within 10 sec.  The endocytosis was independent of the amplitude and duration of depolarization pulses, and linear with the exocytosis.  This result implies that the endocytosis is triggered by the exocytosis.  Although the rapid endocytosis was independent of the parameters of single pulses, activity could modulate its kinetics.  Different from that reported in the neural-muscle junction and calyx synapses, decreasing the stimulation frequency attenuated the endocytosis.  This phenomenon can be mimicked by alteration of the PKA pathway.  Inhibition of endogenous dynamin's function has n effect on the rapid endocytosis and its activity-dependence.  In additional to the Ca2+-indepednet rapid endocytosis, a slow form of endocytosis (in minutes) exists in the DRG neurons.  This form of endocytosis is also linear to the corresponding exocytosis, and is Ca2+-depednet.  Disruption of the cytoskeleton has implication in the endocytosis.  Our results also showed that destroy of the microtube system could nearly abolished the slow endocytosis.  Our results will help the understanding of the mechanisms in the endocytosis, especially in the activity- and PKA-modulation of the endocytosis.

3)        We here describe a novel mechanism for plasma membrane insertion of delta-opioid receptor (DOR).  Activation of the surface DORs causes a slow Ca2+ elevation by both inositol 1, 4, 5-trisphosphate receptor-mediated intracellular Ca2+ release and extracellular Ca2+ entry, resulting in a slow and long-lasting increase in intracellular Ca2+, exocytosis, DOR insertion and CGRP release.  These findings suggest that DOR mediates a distinct positive-feedback mechanism to determine DOR insertion that is coupled with neuropeptide release, and thus reveal an unexpected function for one of the opioid receptors.

  Taken together, our results demonstrated that APs play some important roles in stimulus-secretion coupling.  The depolarization of membrane induces secretion directly without Ca involvement in DRG neurons.  These results help to understand the roles of APs in the stimulus-secretion process, and will also imply that membrane depolarization, as a natural signal in neurons, might has some direct roles in the vesicle recycling except just as a trigger for channels.

  Key borad:  DRG   Calcium   Secretion   Endocytosis   Phosphorylation   PKA   DOR