Pf. Augustijns et Rt. Borchardt, TRANSPORT AND METABOLISM OF DELTA-SLEEP-INDUCING PEPTIDE IN CULTURED HUMAN INTESTINAL EPITHELIAL-CELL MONOLAYERS, Drug metabolism and disposition, 23(12), 1995, pp. 1372-1378
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.