Meredith M, Li G, Metz S A
Department of Medicine, University of Wisconsin and the William S. Middleton Veteran's Administration Hospital, Madison, USA.
Biochem Pharmacol. 1997 Jun 15;53(12):1873-82. doi: 10.1016/s0006-2952(97)00057-9.
Using intact rat islets, we previously observed that GTP depletion (achieved through the use of mycophenolic acid or other synthesis inhibitors) impedes nutrient- but not K+-induced insulin secretion. It was concluded that a proximal nutrient-dependent step in stimulus-secretion coupling (but not the process of Ca2+-induced exocytosis itself) is modulated by ambient GTP levels. To examine Ca2+-dependent steps further in intact beta cells, INS-1 cells (which synthesize GTP and ATP similarly to rat islets) and HIT-T15 cells (whose synthesis of purine nucleotides is different) were studied following cell culture for 1-18 hr in various concentrations of mycophenolic acid (MPA) or mizoribine (MZ). Both agents profoundly reduced GTP content (mean: -78%) and lowered the GTP/GDP ratio by an average of -73%; concomitantly, MPA or MZ reduced insulin secretion induced by 10 mM glucose, 30 or 40 mM KCl, or 100 microM tolbutamide, independent of any changes in cell viability, insulin content, ATP content, the ATP/ADP ratio, or cytosolic free Ca2+ concentrations. In INS-1 cells (which appear to have normal nucleobase transport and "salvage" pathway activities), guanine (but not adenine) restored GTP content, the GTP/GDP ratio, and Ca2+-induced secretion. In HIT cells, the phosphoribosylation of exogenous guanine or hypoxanthine is defective; however, provision of 500 microM guanosine (but not adenosine) reversed the effects of MPA. We conclude that, at least in certain situations, a requisite role for GTP in the distal step(s) of exocytosis can be demonstrated.
我们之前使用完整的大鼠胰岛观察到，GTP耗竭（通过使用霉酚酸或其他合成抑制剂实现）会阻碍营养物质诱导的胰岛素分泌，但不会阻碍钾离子诱导的胰岛素分泌。得出的结论是，刺激-分泌偶联中近端营养物质依赖性步骤（而非钙离子诱导的胞吐作用本身的过程）受细胞外GTP水平调节。为了进一步研究完整β细胞中钙离子依赖性步骤，在不同浓度的霉酚酸（MPA）或咪唑立宾（MZ）中对INS-1细胞（其合成GTP和ATP的方式与大鼠胰岛相似）和HIT-T15细胞（其嘌呤核苷酸合成方式不同）进行1-18小时的细胞培养后进行了研究。两种药物均显著降低了GTP含量（平均降低78%），并使GTP/GDP比值平均降低73%；同时，MPA或MZ降低了由10 mM葡萄糖、30或40 mM氯化钾或100 μM甲苯磺丁脲诱导的胰岛素分泌，且与细胞活力、胰岛素含量、ATP含量、ATP/ADP比值或胞质游离钙离子浓度的任何变化无关。在INS-1细胞（其似乎具有正常的核碱基转运和“补救”途径活性）中，鸟嘌呤（而非腺嘌呤）恢复了GTP含量、GTP/GDP比值以及钙离子诱导的分泌。在HIT细胞中，外源性鸟嘌呤或次黄嘌呤的磷酸核糖基化存在缺陷；然而，提供500 μM鸟苷（而非腺苷）可逆转MPA的作用。我们得出结论，至少在某些情况下，可以证明GTP在胞吐作用的远端步骤中具有必要作用。
