Department of Medicine, State University of New York Upstate Medical University, Syracuse, NY, USA.
J Physiol. 2010 Dec 15;588(Pt 24):4871-89. doi: 10.1113/jphysiol.2010.198424. Epub 2010 Nov 1.
Calcium can be mobilized in pancreatic β-cells via a mechanism of Ca(2+)-induced Ca(2+) release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in β-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C- (PLC-) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC- gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the β-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse β-cells were loaded with the photolabile Ca(2+) chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca(2+) generated CICR in only 9% of the β-cells tested, whereas CICR was generated in 82% of the β-cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A (6-Bnz-cAMP-AM) or Epac2 (8-pCPT-2'-O-Me-cAMP-AM) selectively. However, in β-cells of PLC- KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC- KO β-cells with recombinant PLC- rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC- with a mutated Ras association (RA) domain, or a H1640L PLC- that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT β-cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC- exists in β-cells, and that the activities of Epac2 and PLC- are key determinants of CICR in this cell type.
钙可以通过钙（2+）诱导的钙释放（CICR）机制在胰腺β细胞中动员，并且 cAMP 升高剂（如 exendin-4）通过激活蛋白激酶 A 和 Epac2 来促进 β 细胞中的 CICR。在这里，我们首次报道了一种新型的磷酯酶 C-（PLC-）在胰岛中表达，并且在小鼠中敲除 PLC-基因表达会破坏 exendin-4 的作用，从而促进这些小鼠β细胞中的 CICR。因此，在本研究中，在野生型（WT）C57BL/6 小鼠β细胞中加载光不稳定的 Ca（2+）螯合剂 NP-EGTA 后，UV 闪光光解催化的 Ca（2+）去笼仅在 9%的测试β细胞中产生 CICR，而在用 exendin-4 预处理的β细胞中则产生 82%的 CICR。这种 exendin-4 促进 CICR 的作用可以通过选择性激活蛋白激酶 A（6-Bnz-cAMP-AM）或 Epac2（8-pCPT-2'-O-Me-cAMP-AM）的 cAMP 类似物来复制。然而，在 PLC- KO 小鼠的β细胞中，以及 Epac2 KO 小鼠的β细胞中，这些测试物质在 CICR 测定中表现出不同的效力，使得 exendin-4 部分有效，6-Bnz-cAMP-AM 完全有效，而 8-pCPT-2'-O-Me-cAMP-AM 则没有显著效果。重要的是，用重组 PLC-转导 PLC- KO β细胞可恢复 8-pCPT-2'-O-Me-cAMP-AM 促进 CICR 的作用，而具有突变 Ras 相关（RA）结构域的 K2150E PLC-或催化失活的 H1640L PLC-均无效。由于 8-pCPT-2'-O-Me-cAMP-AM 未能促进转导 GTP 酶激活蛋白（RapGAP）的 WT β细胞中的 CICR，该 RapGAP 下调 Rap 活性，现有证据表明，一种由 Epac2、Rap 和 PLC-组成的信号转导“模块”存在于β细胞中，并且 Epac2 和 PLC-的活性是该细胞类型中 CICR 的关键决定因素。
