Department of Pharmacotherapy, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi, Minami-ku, Hiroshima, Japan.
Br J Pharmacol. 2010 Jan 1;159(2):428-37. doi: 10.1111/j.1476-5381.2009.00544.x. Epub 2009 Dec 15.
Stress on the endoplasmic reticulum (ER) can trigger rescuer responses such as the unfolded protein response (UPR). However, pharmacological modulators of these ER-regulated stress responses are not well understood. In the present study, we found that amiloride, a potassium-sparing diuretic, has unique properties relating to such stress.
We treated mouse primary cultured glial cells with amiloride, in the absence and presence of the ER stress-inducing reagents tunicamycin (Tm) or dithiothreitol, and measured UPR and ER stress-induced cell death. IRE1alpha phosphorylation, eIF2alpha phosphorylation, X-box binding protein 1 (XBP1) splicing, glucose regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) expression by reverse transcription-polymerase chain reaction and Western blotting were used to assess UPR and lactate dehydrogenase activity was determined to measure ER stress-induced cell death.
Amiloride completely inhibited ER stress-induced activation of IRE1alpha, an ER-localized stress sensor protein, splicing of XBP1, and subsequent expression of GRP78 at the mRNA and protein levels. ER stress induces the phosphorylation of eIF2alpha, leading to the expression of CHOP or an attenuation of translation in cells. Surprisingly, treatment with amiloride alone markedly promoted the phosphorylation but actually inhibited ER stress-induced CHOP expression. Finally, we found that amiloride (200 microM) synergistically enhanced ER stress-induced cell death, which was mediated through caspases. On the other hand, a low dose of amiloride (20 microM) significantly prevented Tm-induced cell death.
These results suggest that amiloride can modulate UPR. They also suggest amiloride to be an important pharmacological agent and provide basic information for understanding and preventing ER stress-related diseases.
内质网（ER）所受压力可引发未折叠蛋白反应（UPR）等保护反应。然而，人们对内质网调控应激反应的药理学调节剂知之甚少。本研究发现，保钾利尿剂阿米洛利与应激具有独特关联。
我们用阿米洛利处理原代培养的小鼠神经胶质细胞，分别在无内质网应激诱导剂（衣霉素或二硫苏糖醇）和存在内质网应激诱导剂的情况下进行处理，测量 UPR 和 ER 应激诱导的细胞死亡。通过逆转录-聚合酶链反应和 Western blot 检测 IRE1α磷酸化、eIF2α磷酸化、X 盒结合蛋白 1（XBP1）剪接、葡萄糖调节蛋白 78（GRP78）和 CCAAT/增强子结合蛋白同源蛋白（CHOP）的表达，以评估 UPR；通过测定乳酸脱氢酶活性检测 ER 应激诱导的细胞死亡。
阿米洛利完全抑制了内质网应激诱导的 IRE1α激活、XBP1 的剪接以及 GRP78 在 mRNA 和蛋白水平的表达。内质网应激诱导 eIF2α磷酸化，导致 CHOP 的表达或细胞内翻译减少。令人惊讶的是，单独用阿米洛利处理可显著促进 ER 应激诱导的 CHOP 表达磷酸化，但实际上抑制了 CHOP 的表达。最后，我们发现阿米洛利（200 μM）与 ER 应激诱导的细胞死亡协同增强，这是通过半胱天冬酶介导的。另一方面，低剂量阿米洛利（20 μM）可显著防止 Tm 诱导的细胞死亡。
这些结果表明，阿米洛利可调节 UPR。它们还表明阿米洛利是一种重要的药理学调节剂，为理解和预防与内质网应激相关的疾病提供了基础信息。
