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首页> 外文期刊>Cellular Physiology and Biochemistry >The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells
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The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells

机译：H + / K + ATPase抑制剂SCH-28080抑制INS-1E大鼠胰岛素瘤细胞中胰岛素分泌并诱导细胞死亡

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>Background/Aims: Glucose-stimulated insulin secretion (GSIS) of pancreatic ?2-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem), activation of voltage-activated Ca2+ currents (ICav) and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi) or cell volume, which also affect ?2-cell viability, can elicit or modify insulin release. In ?2-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. Methods: In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi) recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (?”?¨m) were measured by flow cytometry. Results: We found that the ?±-subunit of the non-gastric H+/K+ ATPase (HK?±2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 ?μM SCH-28080 inhibited GSIS by a??50%, while basal release was unaffected. The L-type ICav blocker nifedipine caused a full inhibition of GSIS at 10 and 20 ?μM. At 20 ?μM, SCH-28080 inhibited ICav comparable to 20 ?μM nifedipine and in addition augmented IKATP recorded at -60 mV and hyperpolarized Vmem by a??15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC50 values of 22.9 ?μM and 15.3 ?μM, respectively. At 20 ?μM the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40-50 ?μM, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of ?”?¨m within 4 h under 50 and 100 ?μM while 10 and 20 ?μM had no effect on ?”?¨m within 24 h. Conclusion: We demonstrate expression of HK?±2 in rat INS-1E cells. However, the pump is apparently non-functional under the given conditions. Nonetheless the H+/K+ ATPase blocker SCH-28080 inhibits insulin secretion and induces cell death. Importantly, we show that SCH-28080 inhibits ICav - and activates KATP channels identifying them as novel a€?off-targetsa€? of the inhibitor, causing hyperpolarization of Vmem and inhibition of insulin secretion.
机译：> 背景/目的： 胰岛β2细胞的葡萄糖刺激的胰岛素分泌（GSIS）涉及葡萄糖的摄取和代谢，K ATP的关闭通道和细胞膜电位（V mem ）的去极化，电压激活的Ca 2 + 电流（ICa v ）的激活和Ca 2 + 大量涌入，最终触发激素胞吐作用。除此经典途径外，还可以引起K ATP 独立的机制，例如细胞内pH（pH i ）或细胞体积的变化，这也会影响β2细胞的活力。调节胰岛素释放。在？2细胞中，pH i 的调节主要通过Na + / H + 交换器（NHEs）完成。为了研究除NHEs以外的其他质子挤出机制是否参与pH调节，我们测试了大鼠胰岛素瘤细胞中是否存在非胃H + / K + ATPase。评估了H + / K + ATPase抑制剂SCH-28080对胰岛素分泌，细胞活力和凋亡的影响。 方法： 在INS-1E细胞培养物中，通过以下方法分析H + / K + ATPase基因和蛋白质表达反转录PCR和蛋白质印迹。采用BCECF法通过NH 4 Cl预脉冲法测定急性酸性负荷后细胞内pH（pH i ）的恢复。通过ELISA从细胞培养上清液中测定胰岛素分泌。使用膜片钳记录V mem ，K + 和Ca 2 + 电流。使用刃天青（生存力）和细胞毒性测定法确定总体细胞反应。平均细胞体积（MCV），细胞粒度（侧向散射; SSC），磷脂酰丝氨酸（PS）暴露，细胞膜完整性，半胱天冬酶活性和线粒体膜电位（？”？m m）通过流式细胞仪测量。 结果： 我们发现非胃H + / K + ATPase的？±亚基（ HKα±2）在mRNA和蛋白质水平上表达。但是，与大鼠结肠组织相比，INS-1E细胞中的mRNA丰度非常低。在NH 4 Cl预脉冲实验中，在无Na + 的细胞外条件下未观察到依赖K + 的pH i 恢复条件。尽管如此，在1 h内，20 µμM SCH-28080抑制GSIS达50％，而基础释放不受影响。 L型ICa sub 阻滞剂硝苯地平在10和20μM时可完全抑制GSIS。在20μM时，SCH-28080抑制的ICa v 与20μμM硝苯地平相当，此外在-60 mV和超极化V mem < / sub> 15 mV。 SCH-28080处理后2和24小时的细胞存活率呈剂量依赖性抑制，IC 50 值分别为22.9 µM和15.3 µM。在20？μM时，膜联蛋白V +，胱天蛋白酶+和碘化丙啶+细胞的百分比在24和48小时后显着增加。同时，MCV显着降低（凋亡体积降低，AVD），SSC信号升高。在浓度> 40-50 µμM时，SCH-28080逐渐发生细胞毒性，导致在处理后2小时内坏死细胞急剧增加，在50和100以下的4 h内破坏了β？m m ？μM，而10和20？μM在24小时内对？”？ m 没有影响。 结论： 我们证明了HK？±2在大鼠INS-1E细胞中的表达。但是，泵在给定条件下显然无法正常工作。尽管如此，H + / K + ATPase阻断剂SCH-28080抑制胰岛素分泌并诱导细胞死亡。重要的是，我们表明SCH-28080抑制了ICa v -并激活了K ATP 通道，将其识别为新颖的脱靶酶。抑制剂的作用，导致V mem 超极化并抑制胰岛素分泌。

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