Dziki A J, Batzri S, Harmon J W, Molloy M
Department of Surgery, Veterans Affairs Medical Center, Washington, District of Columbia 20422, USA.
Am J Physiol. 1995 Aug;269(2 Pt 1):G287-96. doi: 10.1152/ajpgi.1995.269.2.G287.
Ca2+ entry into the cell may be an early event in the pathophysiology of bile salt-induced gastric mucosal injury. The aim of this study was to characterize the rise in cytosolic free Ca2+ associated with bile salt injury and its association with cell injury and death. Rabbit gastric mucosal cells were preloaded with the Ca2+ indicator fura 2-acetoxymethyl ester (fura 2-AM) for 20 min at 37 degrees C and then exposed to graded concentrations of the bile salt deoxycholate (DC). Cytosolic free Ca2+ concentration ([Ca2+]i) was estimated by spectrofluorometry. The resting [Ca2+]i in gastric cells was 177 +/- 15 nM (n = 6). When cells were subjected to 0.5 mM DC, there was a time-dependent rise in [Ca2+]i. An increase in [Ca2+]i was observed within 2 min, at which time [Ca2+]i rose from 177 +/- 15 to 480 +/- 30 nM. The maximal increase in [Ca2+]i was observed after 20 min of exposure to 0.5 mM DC (639 +/- 49 nM), and [Ca2+]i remained unchanged for at least 2 h. The increase in [Ca2+]i depended on the concentration of DC. The minimum effective dose of DC was 0.2 mM, with which [Ca2+]i was increased by 1.6-fold (from 177 to 285 nM). At 0.5 mM DC also caused a rise in 45Ca2+ influx into the cells and reduced the viability of gastric cells from 96% to 58% at 2 h. The DC-induced rise in cytosolic free Ca2+ depended on the presence of extracellular Ca2+. In the absence of extracellular Ca2+ there was no rise in cytosolic Ca2+ and gastric cells were protected from cell death caused by DC. The DC-induced cell death was reduced from 26% to 10% and from 37% to 16% at 60 and 90 min, respectively, by removal of extracellular Ca2+. The association of DC with gastric cells was not altered by removing extracellular Ca2+. This suggests decreased DC-induced injury in the absence of extracellular Ca2+ is due to the protection from cellular hypercalcemia rather than some other mechanism related to reduced binding and/or association of DC to gastric cells. These experiments show that rising [Ca2+]i appears to be an early pathophysiological event in bile salt-induced cellular injury and that extracellular Ca2+ is critical to produce this effect.
钙离子进入细胞可能是胆盐诱导胃黏膜损伤病理生理学中的一个早期事件。本研究的目的是描述与胆盐损伤相关的胞质游离钙离子浓度升高情况及其与细胞损伤和死亡的关系。兔胃黏膜细胞在37℃下用钙离子指示剂fura 2 - 乙酰氧甲酯（fura 2 - AM）预加载20分钟，然后暴露于不同浓度的胆盐脱氧胆酸盐（DC）中。通过荧光分光光度法估算胞质游离钙离子浓度（[Ca2+]i）。胃细胞中的静息[Ca2+]i为177±15 nM（n = 6）。当细胞暴露于0.5 mM DC时，[Ca2+]i出现时间依赖性升高。在2分钟内观察到[Ca2+]i增加，此时[Ca2+]i从177±15 nM升至480±30 nM。暴露于0.5 mM DC 20分钟后观察到[Ca2+]i的最大增加（639±49 nM），并且[Ca2+]i至少2小时保持不变。[Ca2+]i的增加取决于DC的浓度。DC的最小有效剂量为0.2 mM，此时[Ca2+]i增加了1.6倍（从177 nM升至285 nM）。0.5 mM DC也导致45Ca2+流入细胞增加，并使胃细胞的活力在2小时内从96%降至58%。DC诱导的胞质游离钙离子浓度升高依赖于细胞外钙离子的存在。在没有细胞外钙离子的情况下，胞质钙离子没有升高，胃细胞免受DC引起的细胞死亡。通过去除细胞外钙离子，DC诱导的细胞死亡在60分钟和90分钟时分别从26%降至10%和从37%降至16%。去除细胞外钙离子不会改变DC与胃细胞的结合。这表明在没有细胞外钙离子的情况下DC诱导的损伤减少是由于免受细胞高钙血症，而不是与DC与胃细胞结合和/或关联减少相关的其他机制。这些实验表明，[Ca2+]i升高似乎是胆盐诱导细胞损伤中的一个早期病理生理事件，并且细胞外钙离子对于产生这种效应至关重要。
