Guest P C, Bailyes E M, Hutton J C
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR, UK.
Biochem J. 1997 Apr 15;323 ( Pt 2)(Pt 2):445-50. doi: 10.1042/bj3230445.
The role of intracellular Ca2+ in the proteolytic processing and intracellular transport of secretory granule proproteins was investigated by pulse-chase radiolabelling of isolated rat islets of Langerhans. The conversion of proinsulin was inhibited by depletion of medium Ca2+ with EGTA and by blocking the transport of Ca2+ into cells with the Ca2+-channel antagonists verapamil, nifedipine and NiCl2. Proinsulin conversion was also reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, indicating that the process requires transport of Ca2+ into the endoplasmic reticulum. This was supported by the finding that proinsulin processing was inhibited when Ca2+ was depleted before or during pulse-labelling, but not after transport of the protein to post-endoplasmic-reticulum compartments. Similarly, the inhibition of proinsulin processing was reversed by re-introduction of medium Ca2+ around the time of radiolabelling, but not after 15 min of chase incubation. Ca2+ depletion also decreased proteolytic maturation of the prohormone convertases PC1, PC2 and carboxypeptidase H. Secretion experiments suggested that the rate and extent of proinsulin transport into secretory granules were inhibited marginally by Ca2+ depletion, whereas those of the convertases were markedly impeded. Inhibition of proinsulin conversion by Ca2+ depletion was thus not simply related to the Ca2+-dependencies of mature PC1 and PC2, but also to a requirement for endoplasmic reticulum Ca2+ in proteolytic maturation of the convertases and in their transfer to secretory granules. The results also suggest that the Ca2+ required for prohormone processing in the granules enters the secretory pathway via the endoplasmic reticulum.
通过对分离的大鼠胰岛进行脉冲追踪放射性标记，研究了细胞内钙离子在分泌颗粒前体蛋白的蛋白水解加工和细胞内运输中的作用。用乙二醇双四乙酸（EGTA）耗尽培养基中的钙离子，以及用钙离子通道拮抗剂维拉帕米、硝苯地平和氯化镍阻断钙离子进入细胞，均可抑制胰岛素原的转化。内质网钙离子ATP酶抑制剂毒胡萝卜素也可降低胰岛素原的转化，这表明该过程需要钙离子转运到内质网中。这一观点得到了以下发现的支持：在脉冲标记前或标记过程中耗尽钙离子时，胰岛素原加工受到抑制，但在蛋白质转运到内质网后区室后则不受影响。同样，在放射性标记时重新引入培养基中的钙离子可逆转胰岛素原加工的抑制作用，但在追踪孵育15分钟后则不能。钙离子耗尽还会降低激素原转化酶PC1、PC2和羧肽酶H的蛋白水解成熟。分泌实验表明，钙离子耗尽对胰岛素原转运到分泌颗粒中的速率和程度有轻微抑制作用，而对转化酶的转运则有明显阻碍。因此，钙离子耗尽对胰岛素原转化的抑制作用不仅与成熟PC1和PC2对钙离子的依赖性有关，还与内质网钙离子在转化酶的蛋白水解成熟及其向分泌颗粒转移中的需求有关。结果还表明，颗粒中激素原加工所需的钙离子通过内质网进入分泌途径。
