Weaver C D, Gundersen V, Verdoorn T A
CNS Drug Discovery, Bristol-Meyers Squibb, Wallingford, Connecticut 06492, USA.
J Biol Chem. 1998 Jan 16;273(3):1647-53. doi: 10.1074/jbc.273.3.1647.
To examine the role of glutamatergic signaling in the function of pancreatic islets, we have characterized a high affinity glutamate/aspartate uptake system in this tissue. The islet [3H]glutamate uptake activity was Na(+)-dependent, and it was blocked by L-trans-pyrrolidine-2,4-dicarboxylic acid, a blocker of neuronal and glial glutamate transporters. Islet glutamate transport activity exhibited a Vmax of 8.48 +/- 1.47 fmol/min/islet (n = 4), which corresponds to 102.2 +/- 17.7 pmol/min/mg islet protein. The apparent Km of islet glutamate transport activity depended on the glucose concentration used in the assay. In the presence of glucose concentrations that do not stimulate insulin secretion (2.8 mM), the apparent Km was 34.7 +/- 7.8 microM (n = 3). However, in high glucose (16.7 mM) the apparent Km increased to 112.7 +/- 16.5 microM (n = 3) with little or no change in Vmax. Like most known plasma membrane glutamate transporters, islet glutamate transporters also transported D-aspartate. Anti-D-aspartate immunoreactivity showed that the islet glutamate/aspartate transport activity was localized to the non-beta cell islet mantle. In perifusion experiments with isolated islets in the absence of exogenous amino acids, L-trans-pyrrolidine-2,4-dicarboxylic acid in the presence of 8.3 mM glucose potentiated insulin secretion 23.3 +/- 2.3% (n = 3) compared with 8.3 mM glucose alone. This effect was abolished in the presence of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione alone inhibited glucose-stimulated insulin secretion in isolated islets by 15.9 +/- 5.9% (n = 3). Taken together these data suggest that a high affinity glutamate transport system exists in pancreatic islets and that this system contributes to a glutamatergic signaling pathway that can modulate glucose-inducible insulin secretion.
为了研究谷氨酸能信号在胰岛功能中的作用，我们对该组织中的一种高亲和力谷氨酸/天冬氨酸摄取系统进行了表征。胰岛的[3H]谷氨酸摄取活性依赖于Na(+)，并被L-反式-脯氨酸-2,4-二羧酸阻断，后者是神经元和胶质细胞谷氨酸转运体的阻断剂。胰岛谷氨酸转运活性的Vmax为8.48±1.47 fmol/分钟/胰岛（n = 4），相当于102.2±17.7 pmol/分钟/毫克胰岛蛋白。胰岛谷氨酸转运活性的表观Km取决于测定中使用的葡萄糖浓度。在不刺激胰岛素分泌的葡萄糖浓度（2.8 mM）存在下，表观Km为34.7±7.8 microM（n = 3）。然而，在高葡萄糖（16.7 mM）条件下，表观Km增加到112.7±16.5 microM（n = 3），而Vmax几乎没有变化。与大多数已知的质膜谷氨酸转运体一样，胰岛谷氨酸转运体也转运D-天冬氨酸。抗D-天冬氨酸免疫反应表明，胰岛谷氨酸/天冬氨酸转运活性定位于非β细胞胰岛被膜。在没有外源氨基酸的情况下对分离的胰岛进行灌流实验，与单独使用8.3 mM葡萄糖相比，在8.3 mM葡萄糖存在下，L-反式-脯氨酸-2,4-二羧酸使胰岛素分泌增强了23.3±2.3%（n = 3）。在存在α-氨基-3-羟基-5-甲基异恶唑-4-丙酸受体拮抗剂6-氰基-7-硝基喹喔啉-2,3-二酮的情况下，这种作用被消除。此外，单独使用6-氰基-7-硝基喹喔啉-2,3-二酮可使分离胰岛中的葡萄糖刺激的胰岛素分泌抑制15.9±5.9%（n = 3）。综合这些数据表明，胰岛中存在一种高亲和力谷氨酸转运系统，并且该系统有助于形成一种可调节葡萄糖诱导的胰岛素分泌的谷氨酸能信号通路。
