胰淀素抑制高糖刺激INS-1细胞内钙离子浓度升高的作用机制

 目的 分析阐明胰淀素短时间作用于INS-1细胞、抑制高糖刺激的细胞内钙离子浓度（［Ca²⁺］_i）升高的机制。方法 采用钙离子荧光指示剂Fluo-4/AM负载细胞后，激光共聚焦显微镜下连续动态观察INS-1细胞经胰淀素孵育前后在葡萄糖、KCl、咖啡因和卡巴胆碱存在条件下［Ca²⁺］_i荧光强度变化。结果 （1）单纯16.7 mmol/L葡萄糖刺激使细胞内钙离子荧光强度变化的曲线下面积（AUC）为990±16；0.5 μmol/L胰淀素使16.7 mmol/L葡萄糖刺激的胞内荧光强度有所下降（AUC为831±10）， 1.0、5.0、10.0 μmol/L胰淀素均可使细胞内荧光强度显著降低（AUC分别为555±9、535±6、531±5），与单纯葡萄糖刺激组比较差异有统计学意义，且呈现剂量依赖趋势。（2）10.0 μmol/L胰淀素可使30 mmol/L KCl刺激的荧光强度明显减低，与单纯KCl刺激组相比（AUC：168±5比311±11）差异有统计学意义。（3） 10.0 μmol/L胰淀素预处理后咖啡因和卡巴胆碱刺激的胞内荧光强度与单纯咖啡因和卡巴胆碱刺激组相比差异无统计学意义。结论 高浓度胰淀素的短时间作用对INS-1细胞经咖啡因和卡巴胆碱刺激的内质网鱼尼丁受体(ryanodine receptor，RyR)、三磷酸肌醇(IP₃)钙库释放没有影响，但可使高糖及KCl刺激的胞内荧光强度降低。推测胰淀素短时间作用使 ［Ca²⁺］_i减少的现象，主要是通过影响膜上钙通道实现的，与胞内钙库的释放无直接相关性。

The mechanism of inhibition of the increase in intracellular calcium concentration by the islet amyloid polypeptide in high glucose-stimulated INS-1 cells

Objective To explore the potential mechanism of the inhibition of increased intracellular free calcium concentration (［Ca²⁺］_i) by short-term exposure to the islet amyloid polypeptide (IAPP) in high glucose-stimulated pancreatic β cells.Methods The pancreatic β cells were loaded with calcium sensitive fluorescent indicator Fluo-4/AM. The fluorescence intensity, which represented ［Ca²⁺］_i, was measured in time by laser scanning confocal microscope before and after stimulated by glucose, KCl, caffeine and carbachol.Results The fluorescence intensity F/F₀ in INS-1 cells, increased to about 2 folds after glucose stimulation. After the exposure to the IAPP with different concentration, the fluorescence intensity F/F₀ was decreased slightly in the pretreated cells by 16.7 mmol/L glucose with 0.5 μmol/L IAPP. However, after the pretreatment of IAPP with the concentration of 1.0, 5.0, 10.0 μmol/L, the fluorescence intensity F/F₀ showed a dose-dependent decrease with statistical difference. The fluorescence intensity F/F₀ in the cells increased rapidly in a peak pattern after the stimulation of 30 mmol/L KCl. But with the pretreatment of 10.0 μmol/L IAPP, the fluorescence intensity F/F₀ decreased with statistical difference. With 20 mmol/L caffeine and 100 μmol/L carbachol which stimulatedCa²⁺ release respectively from internal ryanodine receptor（RYR） and inositol triphosphate （IP₃） Ca²⁺ storage, the fluorescence intensity F/F₀ curve presented a peak pattern. After 10 μmol/L IAPP pretreatment, the fluorescence intensity F/F₀ showed no statistical difference from the control group.Conclusions The short-term effect of IAPP on pancreatic β cells has no influence on the caffeine and carbachol stimulated Ca²⁺ release from endoplasmic reticulum RYR and IP₃ Ca²⁺ storage. The inhibition of calcium increase in INS-1 cells by short-term exposure to IAPP may mainly via inhibiting the voltage-gated L-calcium channels with intact release capacity of Ca²⁺ storage.