Intestinal absorption of β‐lactam antibiotics and oligopeptides

Abstract

The H+‐dependent uptake system responsible for the enteral absorption of oligopeptides and orally active β‐lactam antibiotics was functionally reconstituted into liposomes. Membrane proteins from rabbit small intestinal brush border membrane vesicles were solubilized with n‐octyl glucoside and incorporated into liposomes using a gel filtration method. At protein/lipid ratios of 1:10 and 1:40, the uptake of the orally active α‐amino‐cephalosporin, D‐cephalexin into proteoliposomes was stimulated by an inwardly directed H+ gradient and was protein‐dependent. In these proteoliposomes the binding protein for oligopeptides and β‐lactam antibiotics of Mr 127000 could be labeled by direct photoaffinity labeling with [3H]benzylpenicillin revealing an identical binding specificity as in the original brush border membrane vesicles. The uptake system for β‐lactam antibiotics and oligopeptides showed a remarkable stereospecificity; only D‐cephalexin was taken up by intact brush border membrane vesicles, whereas the L‐enantiomer was not taken up to a significant extent. This stereospecificity for uptake was also seen after reconstitution of solubilized brush border membrane proteins into liposomes demonstrating a functional reconstitution of the peptide transporter. Both enantiomers however, bound to the 127‐kDa binding protein as was shown by a decrease in the extent of photoaffinity labeling of the 127‐kDa protein in the presence of both enantiomers. After reconstitution of subfractions of brush border membrane proteins obtained by wheat germ lectin affinity chromatography into proteoliposomes, only liposomes containing the 127‐kDa binding protein showed a significant uptake of D‐cephalexin whereas the L‐enantiomer was not transported. The uptake rates for D‐cephalexin into proteoliposomes correlated with the content of 127‐kDa binding protein in these liposomes as was determined by specific photoaffinity labeling with [3H]benzylpenicillin. The purified 127‐kDa binding protein was also reconstituted into liposomes and its ability for specific binding of substrates as well as stereospecific uptake of cephalexin could be restored. These results indicate that the binding protein for oligopeptides and β‐lactam antibiotics of Mr 127000 mediates the stereospecific and H+‐dependent transport of orally active β‐lactam antibiotics across the enterocyte brush border membrane. We therefore suggest that this 127‐kDa binding protein is the intestinal peptide transport system (or a component thereof).

Direct Evidence for Efficient Transport and Minimal Metabolism of L-Cephalexin by Oligopeptide Transporter 1 in Budded Baculovirus Fraction

“…19) Whether or not Lcephalexin is transported by PEPT1 has been controversial. One group reported that L-cephalexin was not transported by peptide transporter(s), 17) whereas another group observed substrate-induced intracellular acidification caused by transport of L-cephalexin via a peptide transporter, possibly PEPT1, though they did not detect L-cephalexin itself.…”

“…16) However, intact L-cephalexin was not found in vesicles or proteoliposomes prepared from brush border membrane of intestinal epithelial cells. 17) Although, L-cephalexin was not detected, and the hydrolysis product, 7-aminodesacetoxycephalosporanic acid (7-ADCA), appeared very rapidly in serum and urine after oral administration in rats. 18) This observation suggests that L-cephalexin can be absorbed from gastrointestinal tract.…”

Stereoselective uptake of β‐lactam antibiotics by the intestinal peptide transporter

“…Therefore, a D-configuration in the N-terminus of a peptide substrate does still allow efficient transport. All orally active f-lactams (Amidon & Sinko, 1988; (Ganapathy et al, 1984;Kudo et al, 1989 (Thwaites et al, 1993a, b (Kramer et al, 1992 Since the rabbit small intestinal peptide transporter (PepTI) and the peptide transporter in Caco-2 cells both transport D-as well as L-isomers of aminocephalosporins, it is plausible to conclude that the lack of transport of L-cephalexin in the proteoliposomes containing the 127 kDa protein from rabbit intestine is not caused by species differences of the peptide transporters. As an alternative explanation we could assume that the 127 kDa represents a second transport pathway which, beside PepTl, is responsible for aminocephalosporin transport.…”

“…Previous studies had suggested that L-cephalexin is not transported by the purified brush border membrane peptide transporter from rabbit small intestine when reconstituted into liposomes (Kramer et al, 1992) although L-cephalexin was found to inhibit the uptake of its D-enantiomer into proteoli- Figure 5 Intracellular pH measured in dextran-SNARF-l loaded oocytes expressing the peptide transporter from rabbit small intestine (PepTl). Three days after injection of 5ng of PepTl-cRNA per oocyte, each oocyte was microinjected with the pHsensitive dye dextran-SNARF-l as described in the Methods section.…”

“…Although an interaction of L-isomers of peptides and f-lactam antibiotics with the substrate binding site of the intestinal peptide transporter(s) has been demonstrated by competition experiments in tissue preparations (Matthews & Adibi, 1976;Matthews, 1987;Tamai et al, 1988), brush border membrane vesicles (Rajendran et al, 1985;Tsuji et al, 1986) and Caco-2 cells (Dantzig & Bergin, 1990;Thwaites et al, 1994a) direct evidence for a stereoselective transport of these compounds was absent. In addition, studies with a reconstituted putative peptide transport protein isolated from rabbit small intestine failed to detect any transport of the L-isomer of the aminocephalosporin, cephalexin (Kramer et al, 1992). For these reasons we addressed the question of the stereoselectivity of peptide and fi-lactam transport by investigating whether (a) the cloned rabbit intestinal peptide transporter (PepTl) expressed in Xenopus 1 Author for correspondence.…”