Augustijns P F, Borchardt R T
Department of Pharmaceutical Chemistry, University of Kansas 66045-2504, USA.
Drug Metab Dispos. 1995 Dec;23(12):1372-8.
A cultured human intestinal epithelial (Caco-2) cell monolayer was used to study the transport and metabolism of delta sleep-inducing peptide [DSIP (Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu)]. DSIP is of interest because it has been reported to be capable of permeating biological barriers (e.g. blood-brain barrier), and this property has been related to its solution conformation. When applied to the apical (AP) side of Caco-2 cell monolayers, DSIP was rapidly metabolized (8.2 +/- 1.1% remaining after a 2-hr incubation), affording Trp as the major metabolite and Trp-Ala as a minor metabolite. When DSIP was added to the basolateral (BL) side of the monolayer, the same metabolites were detected, but the peptide was more stable (70.6 +/- 3.0% remaining after a 2-hr incubation). Inclusion of bestatin, an inhibitor of aminopeptidases, at concentrations up to 0.29 mM with DSIP on the AP side of the Caco-2 cell monolayer increased the stability of the peptide only slightly but dramatically altered the distribution of the metabolites (Trp-Ala became the major metabolite, and Trp became the minor metabolite). Inclusion of other aminopeptidase inhibitors (e.g. amastatin, puromycin) alone, dipeptidylpeptidase IV inhibitors (e.g. diprotin A, Gly-Pro) alone, inhibitors of proteases that require heavy metals for proper activity (e.g. EDTA, 1,10-phenanthroline) alone, or cysteine protease inhibitors (e.g. leupeptin) alone did not lead to significant stabilization of the peptide. However, inclusion of a combination of 0.29 mM bestatin and 1 mM diprotin A with DSIP on the AP side of the monolayers resulted in a substantial increase in the stability of the peptide (83.2 +/- 3.7% remaining after a 2-hr incubation). However, under these conditions, a new metabolite (Trp-Ala-Gly-Gly-Asp-Ala-Ser) was observed with a formation that could be inhibited by inclusion of 1 mM captopril, an inhibitor of peptidyl dipeptidase A. Therefore, the stability of DSIP could be further increased (95.1 +/- 1.6% remaining after a 2-hr incubation) by incubating the peptide with 0.29 mM bestatin, 1 mM diprotin A, and 1 mM captopril. However, even when the major metabolic pathways were inhibited on the AP side of the cell monolayer, no DSIP was detected on the BL side of a Caco-2 cell monolayer. These results suggest that a yet unidentified metabolic pathway is preventing the AP-to-BL flux of DSIP or that DSIP has lower "intrinsic" ability to permeate across cultured intestinal epithelial cells than across cultured brain endothelial cells, a cell culture model of the blood-brain barrier.
使用培养的人肠上皮（Caco-2）细胞单层来研究δ睡眠诱导肽[DSIP（色氨酸-丙氨酸-甘氨酸-甘氨酸-天冬氨酸-丙氨酸-丝氨酸-甘氨酸-谷氨酸）]的转运和代谢。DSIP备受关注，因为据报道它能够穿透生物屏障（如血脑屏障），且这种特性与其溶液构象有关。当将DSIP应用于Caco-2细胞单层的顶端（AP）侧时，DSIP迅速代谢（孵育2小时后剩余8.2±1.1%），产生色氨酸作为主要代谢产物，色氨酸-丙氨酸作为次要代谢产物。当将DSIP添加到单层的基底外侧（BL）侧时，检测到相同的代谢产物，但该肽更稳定（孵育2小时后剩余70.6±3.0%）。在Caco-2细胞单层的AP侧，将氨肽酶抑制剂贝司他汀以高达0.29 mM的浓度与DSIP一起加入，仅略微增加了肽的稳定性，但显著改变了代谢产物的分布（色氨酸-丙氨酸成为主要代谢产物，色氨酸成为次要代谢产物）。单独加入其他氨肽酶抑制剂（如抑氨肽酶、嘌呤霉素）、二肽基肽酶IV抑制剂（如二肽素A、甘氨酰-脯氨酸）、需要重金属才能正常发挥活性的蛋白酶抑制剂（如乙二胺四乙酸、1,10-菲咯啉）或半胱氨酸蛋白酶抑制剂（如亮抑酶肽），均未导致肽的显著稳定。然而，在单层的AP侧将0.29 mM贝司他汀和1 mM二肽素A与DSIP一起加入，导致肽的稳定性大幅增加（孵育2小时后剩余83.2±3.7%）。然而，在这些条件下，观察到一种新的代谢产物（色氨酸-丙氨酸-甘氨酸-甘氨酸-天冬氨酸-丙氨酸-丝氨酸），其形成可被加入1 mM的肽基二肽酶A抑制剂卡托普利所抑制。因此，通过将肽与0.29 mM贝司他汀、1 mM二肽素A和1 mM卡托普利一起孵育，DSIP的稳定性可进一步提高（孵育2小时后剩余95.1±1.6%）。然而，即使在细胞单层的AP侧主要代谢途径被抑制时，在Caco-2细胞单层的BL侧也未检测到DSIP。这些结果表明，一种尚未确定的代谢途径正在阻止DSIP从AP侧向BL侧的通量，或者DSIP穿透培养的肠上皮细胞的“内在”能力低于穿透培养的脑内皮细胞（血脑屏障的细胞培养模型）的能力。
