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.
Methods:
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.
Results:
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.
Conclusions:
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.
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