Seward E P, Chernevskaya N I, Nowycky M C
Department of Anatomy and Neurobiology, Medical College of Pennsylvania, Philadelphia 19129, USA.
J Neurosci. 1995 May;15(5 Pt 1):3390-9. doi: 10.1523/JNEUROSCI.15-05-03390.1995.
The link between electrical activity, Ca2+ entry through voltage-gated channels, and transmitter or hormone secretion is a central issue in neurobiology. In peptidergic nerve terminals of the mammalian neurohypophysis (NHP), secretion is elicited by patterned bursts of action potentials (APs). All parameters of the bursts are important to elicit efficient secretion, including AP frequency, AP broadening, burst duration, and interburst interval (Leng, 1988). We have studied Ca(2+)-secretion coupling of peptide-containing large dense-core vesicles (LDCV) in isolated rat NHP terminals. Ca2+ influx through voltage-gated Ca2+ channels was elicited and recorded by the whole-cell patch-clamp technique. Exocytosis was monitored on line with high temporal resolution by the capacitance detection technique (Neher and Marty, 1982). AP bursts were simulated by depolarizing pulse trains that mimic pulsatile submembrane Ca2+ elevations predicted for physiological stimuli. The characteristic capacitance response (delta Cm) to a train of depolarizing pulses was triphasic. It consisted of a threshold phase during which early pulses did not elicit secretion, a subsequent secretory phase during which Cm increases were coupled to depolarizing pulses, and a fatigued or inactivated state during which additional Ca2+ entry was ineffective. Both the threshold phase and secretory phase were correlated with the integrals of Ca2+ current. Ca2+ chelators affect both the threshold and secretory phase at submillimolar concentrations. Thus, a "shell" rather than "microdomain" model of Ca2+ elevation is appropriate for analyzing Ca(2+)-secretion coupling in NHP terminals (Nowycky and Pinter, 1993). We propose a two-step model, with a ca(2+)-dependent preparatory step followed by a final exocytotic step that is coupled to active Ca2+ influx. The results suggest that under physiological conditions, APs early in a burst prepare an NHP terminal for secretion, but later APs actually trigger exocytosis. Since NHP terminals do not possess a readily releasable pool of vesicles that require only a single Ca2+ step for exocytosis as seen in chromaffin cells (Neher and Zucker, 1993) and melanotrophs (Thomas et al, 1993a), Ca(2+)-secretion coupling mechanisms may be heterologous even within a single class of vesicles.
电活动、通过电压门控通道的Ca2+内流与递质或激素分泌之间的联系是神经生物学中的一个核心问题。在哺乳动物神经垂体（NHP）的肽能神经末梢中，动作电位（AP）的模式化爆发引发分泌。爆发的所有参数对于引发高效分泌都很重要，包括AP频率、AP展宽、爆发持续时间和爆发间隔（Leng，1988）。我们研究了分离的大鼠NHP末梢中含肽大致密核心囊泡（LDCV）的Ca（2+）-分泌偶联。通过全细胞膜片钳技术引发并记录通过电压门控Ca2+通道的Ca2+内流。通过电容检测技术（Neher和Marty，1982）以高时间分辨率在线监测胞吐作用。通过模拟生理刺激预测的搏动性膜下Ca2+升高的去极化脉冲序列来模拟AP爆发。对一系列去极化脉冲的特征性电容响应（δCm）是三相的。它包括一个阈值阶段，在此期间早期脉冲不引发分泌；随后是分泌阶段，在此期间Cm的增加与去极化脉冲相关联；以及一个疲劳或失活状态，在此期间额外的Ca2+内流无效。阈值阶段和分泌阶段都与Ca2+电流的积分相关。Ca2+螯合剂在亚毫摩尔浓度下会影响阈值阶段和分泌阶段。因此，Ca2+升高的“壳”模型而非“微区”模型适用于分析NHP末梢中的Ca（2+）-分泌偶联（Nowycky和Pinter，1993）。我们提出了一个两步模型，先是一个依赖Ca（2+）的准备步骤，接着是一个与活跃的Ca2+内流偶联的最终胞吐步骤。结果表明，在生理条件下，爆发早期的AP使NHP末梢为分泌做好准备，但后期的AP实际上触发胞吐作用。由于NHP末梢不像嗜铬细胞（Neher和Zucker，1993）和促黑素细胞（Thomas等人，1993a）那样拥有仅需单个Ca2+步骤即可进行胞吐作用的易于释放的囊泡池，即使在同一类囊泡中，Ca（2+）-分泌偶联机制也可能是异源的。
