Johnston Nikki, Wells Clive W, Samuels Tina L, Blumin Joel H
Department of Otolaryngology and Communication Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Ann Otol Rhinol Laryngol. 2010 Aug;119(8):547-58. doi: 10.1177/000348941011900808.
We undertook to (1) obtain unequivocal evidence to confirm or rebut our initial observations that pepsin is taken up by hypopharyngeal epithelial cells by receptor-mediated endocytosis, (2) investigate whether uptake of pepsin at pH 7, in nonacidic refluxate, is of pathological significance, and 3) test our hypothesis that inactive but stable pepsin (<pH 8) taken up by hypopharyngeal epithelial cells causes damage by becoming reactivated inside the cell.
Human posterior cricoid biopsy specimens and cultured hypopharyngeal FaDu epithelial cells were used to perform competitive binding studies and to investigate colocalization of pepsin with clathrin, Rab-9, and TRG-46. FaDu cells were exposed to pepsin (both irreversibly and reversibly inactivated) in the presence and absence of wortmannin and dimethyl amiloride and analyzed by electron microscopy, MTT cytotoxicity assay, and Stress and Toxicity SuperArray.
Pepsin is unequivocally taken up by hypopharyngeal epithelial cells by receptor-mediated endocytosis. Uptake of pepsin at pH 7, in nonacidic refluxate, causes mitochondrial damage and changes the expression of several genes implicated in stress and toxicity. Irreversible, but not reversible, inhibition of peptic activity prevents these changes.
Pepsin, at pH 7, in nonacidic refluxate, causes damage by becoming reactivated inside the cell. Irreversible inhibitors of peptic activity hold promise as a new therapy for reflux.
我们旨在（1）获取明确证据，以证实或反驳我们最初的观察结果，即胃蛋白酶通过受体介导的内吞作用被下咽上皮细胞摄取；（2）研究在pH 7的非酸性反流物中胃蛋白酶的摄取是否具有病理意义；以及（3）验证我们的假设，即被下咽上皮细胞摄取的无活性但稳定的胃蛋白酶（pH<8）在细胞内重新激活会导致损伤。
使用人环状软骨后活检标本和培养的下咽FaDu上皮细胞进行竞争性结合研究，并研究胃蛋白酶与网格蛋白、Rab-9和TRG-46的共定位。在存在和不存在渥曼青霉素和二甲基amiloride的情况下，将FaDu细胞暴露于胃蛋白酶（不可逆和可逆失活），并通过电子显微镜、MTT细胞毒性试验以及应激和毒性超级阵列进行分析。
胃蛋白酶确实通过受体介导的内吞作用被下咽上皮细胞摄取。在pH 7的非酸性反流物中胃蛋白酶的摄取会导致线粒体损伤，并改变与应激和毒性相关的几个基因的表达。胃蛋白酶活性的不可逆但非可逆抑制可防止这些变化。
在pH 7的非酸性反流物中的胃蛋白酶在细胞内重新激活会导致损伤。胃蛋白酶活性的不可逆抑制剂有望成为一种新的反流治疗方法。
