Matthias Brandsch

Differential Recognition of β-Lactam Antibiotics by Intestinal and Renal Peptide Transporters, PEPT 1 and PEPT 2

This study was initiated to determine if there are differences in the recognition of β-lactam antibiotics as substrates between intestinal and renal peptide transporters, PEPT 1 and PEPT 2. Reverse transcription-coupled polymerase chain reaction and/or Northern blot analysis have established that the human intestinal cell line Caco-2 expresses PEPT 1 but not PEPT 2, whereas the rat proximal tubule cell line SKPT expresses PEPT 2 but not PEPT 1. Detailed kinetic analysis has provided unequivocal evidence for participation of PEPT 2 in SKPT cells in the transport of the dipeptide glycylsarcosine and the aminocephalosporin cephalexin. The substrate recognition pattern of PEPT 1 and PEPT 2 was studied with cefadroxil (a cephalosporin) and cyclacillin (a penicillin) as model substrates for the peptide transporters constitutively expressed in Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). Cyclacillin was 9-fold more potent than cefadroxil in competing with glycylsarcosine for uptake via PEPT 1. In contrast, cefadroxil was 13-fold more potent than cyclacillin in competing with the dipeptide for uptake via PEPT 2. The substrate recognition pattern of PEPT 1 and PEPT 2 was also investigated using cloned human peptide transporters functionally expressed in HeLa cells. Expression of PEPT 1 or PEPT 2 in HeLa cells was found to induce H+-coupled cephalexin uptake in these cells. As was the case with Caco-2 cells and SKPT cells, the uptake of glycylsarcosine induced in HeLa cells by PEPT 1 cDNA and PEPT 2 cDNA was inhibitable by cyclacillin and cefadroxil. Again, the PEPT 1 cDNA-induced dipeptide uptake was inhibited more potently by cyclacillin than by cefadroxil, and the PEPT 2 cDNA-induced dipeptide uptake was inhibited more potently by cefadroxil than by cyclacillin. It is concluded that there are marked differences between the intestinal and renal peptide transporters in the recognition of β-lactam antibiotics as substrates.

Identification of a renal cell line that constitutively expresses the kidney-specific high-affinity H+/peptide cotransporter.

In this study we describe for the first time the identification of a renal cell line that; ex‐presses the kidney‐specific high‐affinity H+/peptide cotransport system. The kidney cell line SKPT‐C193 C1.2 was obtained by SV40 transformation of rat proximal tubular cells. The transport of the dipeptide glycylsarcosine (Gly‐Sar) was studied in this cell line grown as a confluent monolayer on impermeable plastic supports. Uptake of the dipeptide was rapid and was stimulated sixfold by an inwardly directed H+ gradient, with optimal uptake occurring at an extracellular pH of 6.0. The uptake was markedly reduced by the protonophore carbonyl cyanide 4‐ (trifluoromethoxy) phenylhydrazone whether measured at pH 7.5 or 6.0. Intracellular acidification of the cells by NH4CI prepulse also reduced the uptake of glycylsarcosine. The dipeptide uptake was found to be mediated by a high‐affinity transport system with a Michaelis‐Menten constant (Kt) of 67 ± 2 μM and a maximal transport velocity of 1.20 ± 0.02 nmol · 10 min−1 · mg protein−1. Studied over a concentration range of 5 μM to 5 mM, there was no evidence for a second saturable transport component. Di‐ and tripeptides, but not glycine, were strong inhibitors of glycylsarcosine uptake, indicating that these peptides also interact with the transport system with high affinity. Northern blot analysis of poly(A)+RNA from these cells using cDNA probes specific for the human intestinal peptide transporter (PEPT1) or the human kidney‐specific peptide transporter (PEPT2) revealed that the transport system expressed in these cells is PEPT 2. It is concluded that the SKPT‐0193 C1.2 cell line constitutively expresses the kidney‐specific high‐affinity H+/peptide cotransporter described in the proximal tubular epithelial cells of the normal kidney.—Brandsch, M., Brandsch, C., Prasad, P. D., Ganapathy, V., Hopfer, U., Leibach, F. H. Identification of a renal cell line that constitutively expresses the kidney‐specific high‐affinity H+/pep‐ tide cotransporter. FASEBJ. 9, 1489‐1496 (1995)

Influence of proton and essential histidyl residues on the transport kinetics of the H+/peptide cotransport systems in intestine (PEPT 1) and kidney (PEPT 2)

The mechanism by which H+ alters the kinetics of the H+-coupled peptide transporters PEPT 1 and PEPT 2 was investigated in two different cell lines which differentially express these transporters, namely Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). The effects of H+ on the affinity and the maximal velocity of Gly-Sar uptake were analyzed in these cells under identical conditions. In both cells, H+ influenced only the maximal velocity of uptake and not the apparent affinity. The effects of H+ on the IC50 values (i.e., concentration necessary to cause 50% inhibition) of the cationic dipeptide Ala-Lys and the anionic dipeptide Ala-Asp for inhibition of Gly-Sar uptake were also investigated. H+ did not change the IC50 value for Ala-Lys but did decrease the IC50 value for Ala-Asp considerably. The influence of diethylpyrocarbonate (DEP) on the kinetic parameters of PEPT 1 and PEPT 2 was then studied. Histidyl residues are the most likely amino acid residues involved in H+ binding and translocation in H+-coupled transport systems and DEP is known to chemically modify histidyl residues and block their function. DEP treatment altered the maximal velocity of Gly-Sar uptake but had no effect on its K(t) (Michaelis-Menten constant) or the IC50 values of Ala-Lys or Ala-Asp for the inhibition of Gly-Sar uptake. It is concluded that H+ stimulates PEPT 1 and PEPT 2 primarily by increasing the maximal velocity of the transporters with no detectable influence on the substrate affinity.

Calmodulin-dependent modulation of pH sensitivity of the amino acid transport system L in human placental choriocarcinoma cells

The JAR human placental choriocarcinoma cells express the amino acid transport system L. The activity of this system is Na(+)-independent and is stimulated by acidic extracellular pH. Treatment of cells with the calmodulin antagonist CGS 9343B results in a marked stimulation of the system L activity. At a CGS 9343B concentration of 50 microM, the stimulation of activity measured at pH 7.5 is about 75-100%. This effect is not blocked by cycloheximide, actinomycin D, colchicine or cytochalasin D suggesting that the stimulation is not due to de novo synthesis of the carrier protein or recruitment of the carrier protein from an intracellular pool. The stimulatory effect of CGS 9343B is reproducible with other calmodulin antagonists. Treatment with CGS 9343B significantly modifies pH sensitivity of the system. The stimulatory effect of H+ is markedly reduced in treated cells compared to control cells. The stimulation of activity at pH 5.5 vs. pH 7.5 is 55% in control cells but only 8% in treated cells. Similarly, the stimulatory effect of CGS 9343B is reduced by H+. The stimulation of activity seen with 50 microM CGS 9343B is 80% at pH 8.0, but only 26% at pH 5.5. In addition, H+ and CGS 9343B affect the kinetic parameters of system L in a similar manner, the stimulation in both cases being primarily due to an increase in the maximal velocity. The apparent competitive nature between the effects of H+ and CGS 9343B is also observed with other calmodulin antagonists. These results show that the transport function and pH sensitivity of the amino acid transport system L in placental choriocarcinoma cells are modulated by calmodulin by processes which do not involve de novo synthesis nor recruitment of the carrier protein.

Proton-coupled peptide transport in the small intestine and kidney

Carrier-mediated transport of small peptides is a well established phenomenon in the small intestine and kidney (Ganapathy & Leibach, 1986, 1991; Ganapathy et al., 1991; Matthews, 1991; Silbernagl, 1988). In the small intestine, absorption of intact peptides into the enterocytes across the brush border membrane via the peptide transport system plays an important role in the assimilation of dietary proteins. The substrates for the peptide transport system are generated by concerted actions of gastric and pancreatic proteases and brush border peptidases. Of these enzymes, dipeptidylpeptidase IV (DPP IV) and angiotensin converting enzyme (ACE), both of which are associated with the brush border membrane, are of significant importance because they directly generate dipeptides from the amino terminus and the carboxy terminus respectively, of large polypeptides and proteins by sequential action. The enterocytes, in addition, possess highly active cytosolic peptidases which hydrolyze the peptides entering the cell via the brush border membrane peptide transport system. Thus, even though protein digestion products are absorbed into the enterocytes to an appreciable extent in the form of intact peptides, what enters the circulation is predominantly in the form of free amino acids.