Pralong W F, Bartley C, Wollheim C B
Division de Biochimie Clinique, University of Geneva, Switzerland.
EMBO J. 1990 Jan;9(1):53-60. doi: 10.1002/j.1460-2075.1990.tb08079.x.
It is generally believed that the initiation of insulin secretion by nutrient stimuli necessitates the generation of metabolic coupling factors, leading to membrane depolarization and the gating of voltage-sensitive Ca2+ channels. To establish this sequence of events, the kinetics of endogenous fluorescence of reduced pyridine nucleotides [NAD(P)H], reflecting nutrient metabolism, were compared to those of cytosolic calcium ([Ca2+]i) rises in single cultured rat islet beta-cells. In preliminary experiments, the loss of quinacrine fluorescence from prelabelled cells was used as an indicator of secretion. This dye is concentrated in the acidic insulin-containing secretory granules. Both glucose and 2-ketoisocaproate (KIC) raised [Ca2+]i in a dose-dependent manner. There was marked cellular heterogeneity in the [Ca2+]i response patterns. The two nutrient stimuli also increased NAD(P)H fluorescence, again showing cell-to-cell variations. In combined experiments, where the two parameters were measured in the same cell, the elevation of the NAD(P)H fluorescence preceded the rise in [Ca2+]i, confirming the statistical evaluation performed on separate cells. The application of two consecutive glucose challenges revealed coordinated changes in [Ca2+]i and NAD(P)H fluorescence. Finally, quinacrine secretion was stimulated by two nutrients with onset times similar to those recorded for [Ca2+]i elevations. These results clearly demonstrate that increased metabolism occurs during the lag period preceding Ca2+ influx via voltage-sensitive Ca2+ channels, a prerequisite for the triggering of insulin secretion by nutrient stimuli.
一般认为，营养刺激引发胰岛素分泌需要产生代谢偶联因子，导致膜去极化和电压敏感性Ca2+通道的开启。为了确定这一系列事件的顺序，将反映营养代谢的还原吡啶核苷酸[NAD(P)H]的内源性荧光动力学与单个培养的大鼠胰岛β细胞中胞质钙([Ca2+]i)升高的动力学进行了比较。在初步实验中，预先标记细胞中喹吖因荧光的丧失被用作分泌的指标。这种染料集中在含胰岛素的酸性分泌颗粒中。葡萄糖和2-酮异己酸(KIC)均以剂量依赖的方式升高[Ca2+]i。[Ca2+]i反应模式存在明显的细胞异质性。这两种营养刺激也增加了NAD(P)H荧光，同样显示出细胞间的差异。在联合实验中，在同一细胞中测量这两个参数时，NAD(P)H荧光的升高先于[Ca2+]i的升高，证实了对单个细胞进行的统计评估。连续两次葡萄糖刺激的应用揭示了[Ca2+]i和NAD(P)H荧光的协同变化。最后，两种营养物质刺激喹吖因分泌，其起始时间与记录的[Ca2+]i升高的起始时间相似。这些结果清楚地表明，在通过电压敏感性Ca2+通道的Ca2+内流之前的延迟期内发生了代谢增加，这是营养刺激触发胰岛素分泌的先决条件。
