Eto Kazuhiro, Yamashita Tokuyuki, Hirose Kenzo, Tsubamoto Yoshiharu, Ainscow Edward K, Rutter Guy A, Kimura Satoshi, Noda Mitsuhiko, Iino Masamitsu, Kadowaki Takashi
Dept. of Metabolic Diseases, Graduate School of Medicine, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Am J Physiol Endocrinol Metab. 2003 Aug;285(2):E262-71. doi: 10.1152/ajpendo.00542.2002. Epub 2003 Mar 18.
We studied acute changes of secretory vesicle pH in pancreatic beta-cells with a fluorescent pH indicator, lysosensor green DND-189. Fluorescence was decreased by 0.66 +/- 0.10% at 149 +/- 16 s with 22.2 mM glucose stimulation, indicating that vesicular pH was alkalinized by approximately 0.016 unit. Glucose-responsive pH increase was observed when cytosolic Ca2+ influx was blocked but disappeared when an inhibitor of glycolysis or mitochondrial ATP synthase was present. Glutamate dimethyl ester (GME), a plasma membrane-permeable analog of glutamate, potentiated glucose-stimulated insulin secretion at 5 mM without changing cellular ATP content or cytosolic Ca2+ concentration ([Ca2+]). Application of GME at basal glucose concentration decreased DND-189 fluorescence by 0.83 +/- 0.19% at 38 +/- 2 s. These results indicated that the acutely alkalinizing effect of glucose on beta-cell secretory vesicle pH was dependent on glucose metabolism but independent of modulations of cytosolic [Ca2+]. Moreover, glutamate derived from glucose may be one of the mediators of this alkalinizing effect of glucose, which may have potential relevance to the alteration of secretory function by glutamate.
我们使用荧光pH指示剂溶酶体传感器绿色DND-189研究了胰腺β细胞中分泌囊泡pH的急性变化。在22.2 mM葡萄糖刺激下，荧光在149±16秒时下降了0.66±0.10%，表明囊泡pH值碱化了约0.016个单位。当胞质Ca2+内流被阻断时观察到葡萄糖反应性pH升高，但当存在糖酵解抑制剂或线粒体ATP合酶时消失。谷氨酸二甲酯（GME）是一种可透过质膜的谷氨酸类似物，在5 mM时增强了葡萄糖刺激的胰岛素分泌，而不改变细胞ATP含量或胞质Ca2+浓度（[Ca2+]）。在基础葡萄糖浓度下应用GME，在38±2秒时使DND-189荧光下降了0.83±0.19%。这些结果表明，葡萄糖对β细胞分泌囊泡pH的急性碱化作用依赖于葡萄糖代谢，但与胞质[Ca2+]的调节无关。此外，来自葡萄糖的谷氨酸可能是葡萄糖这种碱化作用的介质之一，这可能与谷氨酸对分泌功能的改变具有潜在相关性。
