| 摘要: |
| 首次应用膜片钳技术, 通过监测细胞膜电容变化的方法实时测定去极化电刺激诱导的单个中华绒螯蟹眼柄神经内分泌细胞释放甲壳动物高糖激素(CHH)的胞吐分泌活动。结果表明, 膜电容检测法适用于中华绒螯蟹眼柄神经内分泌细胞胞吐分泌活动的监测, 在细胞的分泌过程中清晰地记录到膜电容的增加。膜电容的增加与去极化时间相关。去极化20ms, 细胞的膜电容开始增加；220ms的去极化诱导细胞分泌CHH达到饱和, 膜电容增加约(521±36)fF(n= 5)。不同幅值去极化电压诱导细胞分泌活动监测结果表明, 只有当去极化电压超过钙离子通道激活阈值才会引起细胞膜电容的增加。这一结果提示中华绒螯蟹眼柄神经肽类激素为细胞外钙离子依赖性分泌。
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| 关键词: 中华绒螯蟹,神经内分泌细胞,分泌活动,膜电容,实时监测 |
| DOI：10.11693/hyhz200305010010 |
| 分类号: |
| 基金项目:国家自然科学基金资助项目,30271019号;国家重点基础研究发展规划(973)资助项目,G1999012007号 |
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| REAL-TIME MONITOR OF SECRETION ACTIVITY IN NEUROSECRETORY CELLS IN THE EYESTALK OF CHINESE MITTEN-HANDED CRABS ERIOCHEIR SINENSIS |
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SUN Jin-Sheng1,2, GAO Chun-Lei1, XIANG Jian-Hai1
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1.Institute of Oceanology, The Chinese Academy of Sciences;2.College of Chemistry and Life Sciences, Tianjin Normal University
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| Abstract: |
| The X-organ-sinus gland (XO-SG) complex is an important neurosecretory organ in Crustacean, analogous to vertebrate hypothalamo-neurohypophyseal system. Some peptide hormones are produced and secreted in the complex, including Crustacean hyperglycemia hormone (CHH), Molt-inhibiting hormone (MIH), Gonad-inhibiting hormone (GIH), Mandibular organ-inhibiting hormone (MOIH) etc. These neuropeptides play a key role in the regulation of a variety of important physiological processes, such as molting, reproduction, carbohydrate metabolism, pigment migration, and osmoregulation. Therefore, XO-SG complex has received most attention all the time. The structure and function of the XO-SG complex are well known in many crustaceans at present. The purification, amino acid analysis and molecular biological aspects of the neuropeptides were also well studied. Due to the deficiency of detecting methods of the
neurohormones, the studies on the neurosecretory regulation of the neuropeptides were already limited in intact animal or acutely isolated optic ganglion preparation. Little knowledge was obtained on the mechanism of stimulation-secretion coupling and neurosecretory regulation till now.
Chinese mitten-handed crab (Eriocheir sinensis) is one of the most important economical valuable crustacean in China. Immunocytochemical localization and immunobloting showed that the anti-CHH immunoreactive cells were located on the proximal surface of X-organ. The secretion activity that was induced by stimulation of depolar voltage was real-time monitored by use of whole-cell patch clamp technique combined with membrane capacitance measurement in the single cell about 2h after plating. The results suggested that th emembrane capacitance measurement hold true in monitoring secretion activity of the neurosecretory cells in the MTXO of Chinese mitten-handed crabs. The membrane capacitance increase was related to the stimulating duration of depolar voltage. The secretion activity monitored by membrane capacitance increase was induced in the duration that exceeded 20ms. The maximal membrane capacitance increase (521±36)fF(n= 5) was observed in the duration of 220ms. The monitoring results of secretion activity induced by the stimulation of different depolar voltage showed that the membrane capacitance increase was positive related to the size of the depolar voltage. Only the depolar voltage that exceeded activating threshold of calcium channel could evoke the secretory activation. This implied that the secretion activity in the neurosecretory cells depended on the entry through the calcium channel expressed in the cell membrane.
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| Key words: Eriocheir sinensis, Neurosecretory cell, Secretionactivity, Membrane capacitance, Real-time monitoring |
