Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
Gastroenterology. 2012 Sep;143(3):832-843.e7. doi: 10.1053/j.gastro.2012.06.011. Epub 2012 Jun 15.
BACKGROUND & AIMS: During development of alcoholic pancreatitis, oxidative (acetaldehyde) and nonoxidative metabolites (ethyl palmitate, ethyl oleate), rather than ethanol itself, mediate toxic injury. Exposure of pancreatic acini to ethanol blocks cholecystokinin (CCK)-8-stimulated apical exocytosis and redirects exocytosis to the basolateral plasma membrane, causing interstitial pancreatitis. We examined how each ethanol metabolite contributes to these changes in exocytosis.
Rat pancreatic acini were incubated with concentrations of ethanol associated with alcoholic pancreatitis (20-50 mmol/L) or ethanol metabolites (1-3 mmol/L) and then stimulated with CCK-8. We performed single zymogen granule (ZG) exocytosis assays, Ca(2+) imaging studies, ultrastructural analyses (with electron microscopy), and confocal microscopy to assess the actin cytoskeleton and track the movement of vesicle-associated membrane protein (VAMP)-8-containing ZGs. Coimmunoprecipitation assays were used to identify complexes that contain the distinct combinations of Munc18 and the soluble N-ethylmaleimide sensitive factor attachment protein receptor proteins, which mediate apical (ZG-apical plasma membrane) and basolateral exocytosis and fusion between ZGs (ZG-ZG).
The ethanol metabolites acetaldehyde, ethyl palmitate, and ethyl oleate reduced CCK-8-stimulated apical exocytosis and formation of apical exocytotic complexes (between Munc18b and Syntaxin-2, synaptosomal-associated protein of 23 kilodaltons [SNAP23], and VAMP2) in rat pancreatic acini. Acetaldehyde and ethyl oleate redirected CCK-8-stimulated exocytosis to the basal and lateral plasma membranes and translocation of VAMP8-containing ZGs toward the basolateral plasma membrane. This process was mediated primarily via formation of basolateral exocytotic complexes (between Munc18c and Syntaxin-4, SNAP23, and VAMP8). Exposure of the acini to acetaldehyde and ethyl oleate followed by CCK-8 stimulation mildly perturbed the actin cytoskeleton and Ca(2+) signaling; exposure to ethyl palmitate severely affected Ca(2+) signaling. Acetaldehyde, like ethanol, promoted fusion between ZGs by the formation of ZG-ZG exocytotic complexes (between Munc18b and Syntaxin-3, SNAP23, and VAMP8), whereas ethyl palmitate and ethyl oleate reduced ZG-ZG fusion and formation of these complexes.
The ethanol metabolites acetaldehyde, ethyl palmitate, and ethyl oleate perturb exocytosis processes in cultured rat pancreatic acini (apical blockade, basolateral exocytosis, and fusion between ZGs). Acetaldehyde and, to a lesser degree, ethyl oleate produce many of the same pathologic effects of ethanol on CCK-8-stimulated exocytosis in pancreatic acini.
在酒精性胰腺炎的发展过程中，氧化（乙醛）和非氧化代谢物（棕榈酸乙酯、油酸乙酯）而不是乙醇本身介导毒性损伤。胰腺腺泡暴露于乙醇会阻断胆囊收缩素（CCK）-8 刺激的顶端胞吐作用，并将胞吐作用重新定向到基底外侧质膜，导致间质胰腺炎。我们研究了每种乙醇代谢物如何导致这些胞吐作用的变化。
用与酒精性胰腺炎相关的乙醇浓度（20-50mmol/L）或乙醇代谢物（1-3mmol/L）孵育大鼠胰腺腺泡，然后用 CCK-8 刺激。我们进行了单个酶原颗粒（ZG）胞吐作用测定、Ca（2+）成像研究、超微结构分析（电子显微镜）和共聚焦显微镜以评估肌动蛋白细胞骨架并追踪含有囊泡相关膜蛋白（VAMP）-8 的 ZG 的运动。共免疫沉淀测定用于鉴定包含不同组合的 Munc18 和可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体蛋白的复合物，这些复合物介导顶端（ZG-顶端质膜）和基底外侧胞吐作用以及 ZG 之间的融合（ZG-ZG）。
乙醇代谢物乙醛、棕榈酸乙酯和油酸乙酯减少了大鼠胰腺腺泡中 CCK-8 刺激的顶端胞吐作用和顶端胞吐作用复合物（Munc18b 与Syntaxin-2、突触相关蛋白 23kDa [SNAP23] 和 VAMP2 之间）的形成。乙醛和油酸乙酯将 CCK-8 刺激的胞吐作用重新定向到基底和侧质膜，并将含有 VAMP8 的 ZG 向基底外侧质膜转移。这个过程主要是通过形成基底外侧胞吐作用复合物（Munc18c 与 Syntaxin-4、SNAP23 和 VAMP8 之间）介导的。用乙醛和油酸酯孵育胰腺腺泡，然后用 CCK-8 刺激，轻微扰乱肌动蛋白细胞骨架和 Ca（2+）信号；用棕榈酸乙酯孵育严重影响 Ca（2+）信号。乙醛像乙醇一样，通过形成 ZG-ZG 胞吐作用复合物（Munc18b 与 Syntaxin-3、SNAP23 和 VAMP8 之间）促进 ZG 之间的融合，而棕榈酸乙酯和油酸乙酯减少 ZG-ZG 融合和这些复合物的形成。
乙醇代谢物乙醛、棕榈酸乙酯和油酸乙酯扰乱了培养的大鼠胰腺腺泡中的胞吐作用过程（顶端阻断、基底外侧胞吐作用和 ZG 之间的融合）。乙醛和在较小程度上，油酸乙酯产生了乙醇对胰腺腺泡中 CCK-8 刺激的胞吐作用的许多相同的病理影响。
