Parnaud Géraldine, Hammar Eva, Rouiller Dominique G, Bosco Domenico
Dept. of Genetic Medicine and Development, Univ. Medical Center, 1 rue Michel Servet, 1211 Geneva 4, Switzerland.
Am J Physiol Endocrinol Metab. 2005 Aug;289(2):E313-21. doi: 10.1152/ajpendo.00006.2005. Epub 2005 Mar 22.
In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca(2+)]i. Finally, calpeptin was shown to affect the [Ca(2+)]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic beta-cells. It is therefore suggested that calpain could be a mediator of Ca(2+)-induced-insulin secretion and beta-cell spreading.
除了促进胰岛素分泌外，葡萄糖引发的胞质Ca(2+)增加已被证明对于附着在细胞外基质（804G基质）上的β细胞铺展至关重要。钙蛋白酶是依赖Ca(2+)的半胱氨酸蛋白酶，参与包括细胞骨架重排和囊泡运输在内的广泛细胞反应。本研究旨在评估钙蛋白酶是否也参与Ca(2+)诱导的大鼠胰腺β细胞胰岛素分泌和铺展过程。结果表明β细胞在804G基质上的铺展依赖于钙蛋白酶。事实上，用三种不同的钙蛋白酶抑制剂（N-乙酰亮氨酰亮氨酰正亮氨酸、钙蛋白酶抑制素和乙基(+)-(2S,3S)-3-[(S)-3-甲基-1-(3-甲基丁基氨基甲酰基)丁基-氨基甲酰基]-2-氧代环丙烷羧酸盐）处理可抑制葡萄糖和氯化钾诱导的细胞铺展，而细胞附着没有明显改变。钙蛋白酶抑制剂还抑制了葡萄糖和氯化钾刺激的胰岛素分泌，而不影响胰岛素合成。将抑制剂从细胞培养物中洗脱24小时后，恢复了在804G基质上的铺展和胰岛素分泌反应。此外，用钙蛋白酶抑制素孵育并不影响对作用于细胞内钙[Ca(2+)]i下游胞吐作用的马斯托帕兰的胰岛素分泌反应。最后，钙蛋白酶抑制素被证明影响对葡萄糖的[Ca(2+)]i反应，但不影响对氯化钾的反应。总之，结果表明钙蛋白酶抑制可阻断原代胰腺β细胞的铺展和胰岛素分泌。因此，提示钙蛋白酶可能是Ca(2+)诱导的胰岛素分泌和β细胞铺展的介质。
