EXOCYTOSIS ELICITED BY ACTION-POTENTIALS AND VOLTAGE-CLAMP CALCIUM CURRENTS IN INDIVIDUAL MOUSE PANCREATIC B-CELLS
EXOCYTOSIS ELICITED BY ACTION-POTENTIALS AND VOLTAGE-CLAMP CALCIUM CURRENTS IN INDIVIDUAL MOUSE PANCREATIC B-CELLS
复制标题
DOI：
10.1113/jphysiol.1993.sp019966
复制
发表时间：
1993-12-01
影响因子：
5.5
通讯作者：
RORSMAN, P
中科院分区：
文献类型：
--
作者：
AMMALA, C;ELIASSON, L;RORSMAN, P
1.结果：1.通过测量细胞膜电容（作为胞吐的指标）和细胞内Ca 2+浓度（[Ca 2 +]i）来确定单个胰腺B细胞分泌的Ca 2+依赖性。胞吐作用依赖于[Ca 2 +]i的升高，并可通过激活电压依赖性Ca 2+电流来诱发。去极化诱导释放的阈值为0.5 μ M [Ca 2 +]i。一旦超过[Ca 2 +]i阈值，胞吐作用迅速（< 50 ms）启动。当施加单独的脉冲时，胞吐在复极化后立即停止，并且Ca 2+通道关闭，尽管[Ca 2 +]i保持升高数秒。在重复刺激（1 Hz）期间，当[Ca 2 +]i达到微摩尔水平时，胞吐作用也在脉冲间隔期间发生，尽管速率比去极化期间慢.胞吐作用可由模拟动作电位启动。单个动作电位仅产生小的电容增加，并且在某些细胞中甚至不能刺激释放，而大于或等于四个动作电位获得更大和更一致的响应。比较胞吐率测量响应去极化，动员的Ca 2+从细胞内存储或输液的Ca 2+通过贴片移液管表明，[Ca 2 +]i在分泌部位达到几个微摩尔浓度。这比通过显微荧光法检测的平均[Ca 2 +]i高得多，表明B细胞内存在陡峭的[Ca 2 +]i空间梯度。在细胞内溶液中加入Ca 2 +/钙调素依赖性蛋白激酶II抑制剂可降低去极化诱导的胞吐反应，表明该酶可能参与[Ca 2 +]i升高与刺激分泌机制之间的偶联。单一胞吐事件的大小为2 fF，对应于250 nm的分泌颗粒直径。8.在短时间内，胞吐作用可能非常快（1 pF/s或500颗粒/s），这比胞吞作用的速率（18 fF/s或9颗粒/s）高得多。由于后者与胰岛分泌胰岛素的最大速率（几乎等于2个颗粒/秒）更一致，我们认为膜修复可能会在长时间的分泌过程中对胞吐速率设置上限。
1. Measurements of membrane capacitance, as an indicator of exocytosis, and intracellular Ca2+ concentration ([Ca2+]i) were used to determine the Ca2+ dependence of secretion in single pancreatic B-cells.2. Exocytosis was dependent on a rise in [Ca2+]i and could be evoked by activation of voltage-dependent Ca2+ currents. The threshold for depolarization-induced release was 0.5 muM [Ca2+]i. Once the [Ca2+]i threshold was exceeded, exocytosis was rapidly (< 50 ms) initiated. When individual pulses were applied, exocytosis stopped immediately upon repolarization and the Ca2+ channels closed, although [Ca2+]i remained elevated for several seconds.3. During repetitive stimulation (1 Hz), when [Ca2+]i attained micromolar levels, exocytosis also took place during the interpulse intervals albeit at a slower rate than during the depolarizations.4. Exocytosis could be initiated by simulated action potentials. Whereas a single action potential only produced a small capacitance increase, and in some cells even failed to stimulate release, larger and more consistent responses were obtained with greater-than-or-equal-to four action potentials.5. Comparison of the rates of exocytosis measured in response to depolarization, mobilization of Ca2+ from intracellular stores or infusion of Ca2+ through the patch pipette suggests that [Ca2+]i at the secretory sites attains a concentration of several micromolar. This is much higher than the average [Ca2+]i detected by microfluorimetry suggesting the existence of steep spatial gradients of [Ca2+]i within the B-cell.6. Inclusion of inhibitors of Ca2+/calmodulin-dependent protein kinase II in the intracellular solution reduced the depolarization-induced exocytotic responses suggesting this enzyme may be involved in the coupling between elevation of [Ca2+]i to stimulation of the secretory machinery.7. The size of the unitary exocytotic event was 2 fF, corresponding to a secretory granule diameter of 250 nm.8. Over short periods, exocytosis may be extremely fast (1 pF/s or 500 granules/s), which is much higher than the rate of endocytosis (18 fF/s or 9 granules/s). Since the latter is in better agreement with the maximum rate of insulin secretion from islets (almost-equal-to 2 granules/s), we suggest that membrane retrieval may set an upper limit on the rate of exocytosis during extended periods of secretion.