title: Transport and metabolism of delta sleep-inducing peptide in cultured human intestinal epithelial cell monolayers.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas 66045-2504, USA.
authors.forename: P F
authors.initials: PF
authors.lastname: Augustijns
authors.forename: R T
authors.initials: RT
authors.lastname: Borchardt
mesh_terms: D000595:Amino Acid Sequence; D000963:Antimetabolites / Q000494:pharmacology; D001692:Biological Transport; D018938:Caco-2 Cells; D002851:Chromatography, High Pressure Liquid; D003701:Delta Sleep-Inducing Peptide / Q000378:metabolism*; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D013696:Temperature
chemical_list: D000963:Antimetabolites; D011480:Protease Inhibitors; D003701:Delta Sleep-Inducing Peptide; D007930:Leucine; C012211:ubenimex