Bai J Z, Saafi E L, Zhang S, Cooper G J
School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Biochem J. 1999 Oct 1;343 Pt 1(Pt 1):53-61.
The objective of these studies was to clarify the role of Ca(2+) in the mechanism of death evoked by human amylin (hA) in islet beta-cells. hA forms fibrils in vitro and islet amyloid in vivo. Here we show that pure synthetic hA aggregated in solution, formed fibrils and evoked death in cultured RINm5F islet beta-cells in a time-dependent (0-24 h) and concentration-dependent (0-20 microM) manner. Dying cells underwent shrinkage of the nucleus, with clumping and segregation of chromatin into masses that lay against the nuclear envelope, and internucleosomal DNA fragmentation. These cells therefore show many features of apoptosis, although aspects of the morphology might be characteristic of this particular cell type rather than of a general apoptotic nature. Aurintricarboxylic acid, an inhibitor of both Ca(2+)-dependent and Ca(2+)-independent nucleases, suppressed this DNA fragmentation and inhibited apoptosis at concentrations between 25 and 200 microM. Direct measurements of the cytoplasmic free Ca(2+) concentration (Ca(2+)) in fura-2 acetoxymethyl ester (AM)-loaded beta-cells showed that neither hA nor its non-cytotoxic homologue, rat amylin were effective in raising Ca(2+). Modulators of Ca(2+) regulation were tested for their effects on hA-induced beta-cell apoptosis. Ca(2+) ionophore (A23187) and thapsigargin (an inhibitor of endoplasmic reticular Ca(2+)-ATPase activity) by themselves evoked apoptosis accompanied by increased Ca(2+). Neither the Ca(2+) channel blocker verapamil, the extracellular Ca(2+) chelator EGTA nor the cytosolic Ca(2+) buffer bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid ('BAPTA')/AM protected beta-cells from hA-evoked apoptosis. Prolonged incubation of beta-cells with a lethal dose of hA altered neither the basal Ca(2+) nor the thapsigargin-induced release of Ca(2+) from intracellular stores. Furthermore, (45)CaCl(2) uptake by RINm5F cells did not differ in the presence or absence of hA. These results suggest that, whereas alterations in cytosolic Ca(2+) homoeostasis do have a significant role in certain forms of beta-cell death, they do not contribute to the pathway of apoptosis evoked by hA in islet beta-cells.
这些研究的目的是阐明钙离子（Ca(2+)）在人胰岛淀粉样多肽（hA）诱发胰岛β细胞死亡机制中的作用。hA在体外形成纤维，在体内形成胰岛淀粉样蛋白。在此我们表明，纯合成的hA在溶液中聚集，形成纤维，并以时间依赖性（0 - 24小时）和浓度依赖性（0 - 20微摩尔）的方式诱发培养的RINm5F胰岛β细胞死亡。濒死细胞的细胞核发生皱缩，染色质凝聚并分离成块状贴靠在核膜上，同时出现核小体间DNA片段化。因此，这些细胞表现出许多凋亡特征，尽管形态学方面的特征可能是这种特定细胞类型所特有的，而非普遍的凋亡性质。金精三羧酸是一种依赖Ca(2+)和不依赖Ca(2+)的核酸酶的抑制剂，在25至200微摩尔的浓度下可抑制这种DNA片段化并抑制凋亡。用fura - 2乙酰氧甲酯（AM）负载的β细胞直接测量细胞质游离Ca(2+)浓度（Ca(2+)）表明，hA及其无细胞毒性的同源物大鼠胰岛淀粉样多肽均不能有效提高Ca(2+)。测试了Ca(2+)调节因子对hA诱导的β细胞凋亡的影响。Ca(2+)离子载体（A23187）和毒胡萝卜素（内质网Ca(2+)-ATP酶活性抑制剂）自身可诱发凋亡并伴有Ca(2+)升高。Ca(2+)通道阻滞剂维拉帕米、细胞外Ca(2+)螯合剂乙二醇双四乙酸（EGTA）以及细胞质Ca(2+)缓冲剂双（邻氨基苯氧基）乙烷 - N,N,N',N'-四乙酸（“BAPTA”）/AM均不能保护β细胞免受hA诱发的凋亡。用致死剂量的hA长时间孵育β细胞，既未改变基础Ca(2+)，也未改变毒胡萝卜素诱导的细胞内钙库中Ca(2+)的释放。此外，有无hA存在时，RINm5F细胞对（45）CaCl(2)的摄取并无差异。这些结果表明，虽然细胞质Ca(2+)稳态的改变在某些形式的β细胞死亡中确实起重要作用，但它们并不参与hA诱发胰岛β细胞凋亡的途径。
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