TRANSPORT AND METABOLISM OF DELTA-SLEEP-INDUCING PEPTIDE IN CULTURED HUMAN INTESTINAL EPITHELIAL-CELL MONOLAYERS

A cultured human intestinal epithelial (Caco-2) cell monolayer was use d to study the transport and metabolism of delta sleep-inducing peptid e [DSIP (Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu)]. DSIP is of interest be cause it has been reported to be capable of permeating biological barr iers (e.g. blood-brain barrier), and this property has been related to its solution conformation, When applied to the apical (AP) side of Ca co-2 cell monolayers, DSIP was rapidly metabolized (8.2 +/- 1.1% remai ning after a 2-hr incubation), affording Trp as the major metabolite a nd Trp-Ala as a minor metabolite. When DSIP was added to the basolater al (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 inc ubation), Inclusion of bestatin, an inhibitor of aminopeptidases, at c oncentrations up to 0.29 mM with DSIP on the AP side of the Caco-2 cel l monolayer increased the stability of the peptide only slightly but d ramatically altered the distribution of the metabolites (Trp-Ala becam e the major metabolite, and Trp became the minor metabolite). Inclusio n 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 inhibitor s (e.g. leupeptin) alone did not lead to significant stabilization of the peptide, However, inclusion of a combination of 0.29 mM bestatin a nd 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 condition s, 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 i nhibitor of peptidyl dipeptidase A, Therefore, the stability of DSIP c ould be further increased (95.1 +/- 1.6% remaining after a 2-hr incuba tion) 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 detec ted on the BL side of a Caco-2 cell monolayer, These results suggest t hat a yet unidentified metabolic pathway is preventing the AP-to-BL fl ux of DSIP or that DSIP has lower ''intrinsic'' ability to permeate ac ross cultured intestinal epithelial cells than across cultured brain e ndothelial cells, a cell culture model of the blood-brain barrier.