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Frederick H Leibach - One of the best experts on this subject based on the ideXlab platform.

  • transport of valganciclovir a ganciclovir prodrug via Peptide transporters pept1 and pept2
    Journal of Pharmaceutical Sciences, 2000
    Co-Authors: Mitsuru Sugawara, Frederick H Leibach, Vadivel Ganapathy, Wei Huang, Youjun Fei, Malliga E Ganapathy
    Abstract:

    In clinical trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal Peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different experimental systems. We also studied the interaction of these two compounds with the Renal Peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with K(i) values (inhibition constant) of 1.68+/-0.30 and 0.043+/- 0.005 mM, respectively. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiological approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal Peptide transporter PEPT1. This prodrug is also recognized by the Renal Peptide transporter PEPT2 with high affinity.

  • valacyclovir a substrate for the intestinal and Renal Peptide transporters pept1 and pept2
    Biochemical and Biophysical Research Communications, 1998
    Co-Authors: Malliga E Ganapathy, Vadivel Ganapathy, Wei Huang, Hong Wang, Frederick H Leibach
    Abstract:

    Valacyclovir is a prodrug of the antiviral agent acyclovir and it does not contain a Peptide bond in its structure. We studied the interaction of valacyclovir with the Peptide transporters in the human intestinal cell line Caco-2 and the rat kidney proximal tubular cell line SKPT which differentially express Peptide transporters PEPT1 and PEPT2. The results of the studies done with these cell lines were confirmed with the cloned Peptide transporters human PEPT1 and rat PEPT2, expressed heterologously in HeLa cells. The activity of the Peptide transporters was assessed by measuring the uptake of radiolabeled glycylsarcosine in the presence of a H+ gradient. Valacyclovir inhibited the uptake of glycylsarcosine with an inhibition constant (Ki) of 0.49 +/- 0.04 mM in Caco-2 cells and 0.17 +/- 0.01 mM in SKPT cells. In both cell types, the inhibition was competitive. Acyclovir, in contrast to valacyclovir, did not interact with the Peptide transporters. Similar results were obtained with heterologously expressed human PEPT1 and rat PEPT2. Valacyclovir inhibited the hPEPT1-mediated glycylsarcosine transport competitively with a Ki value of 0.74 +/- 0.14 mM. The rPEPT2-mediated transport of glycylsarcosine was also inhibited by valacyclovir competitively and the Ki value for the process was 0.39 +/- 0.03 mM. Acyclovir did not interact with either of these cloned Peptide transporters. We conclude that valacyclovir is a substrate for the Peptide transporters PEPT1 and PEPT2 and that a Peptide bond is not a prerequisite for recognition as a substrate by the Peptide transporters.

  • interaction of anionic cephalosporins with the intestinal and Renal Peptide transporters pept 1 and pept 2
    Biochimica et Biophysica Acta, 1997
    Co-Authors: Malliga E Ganapathy, Vadivel Ganapathy, Puttur D Prasad, Bryan Mackenzie, Frederick H Leibach
    Abstract:

    The present study was undertaken to investigate the interaction of anionic cephalosporins (cefixime, ceftibuten, and cefdinir) with the Renal Peptide transporter (PEPT 2) and the intestinal Peptide transporter (PEPT 1) using four different experimental model systems. In the first approach, the human colon carcinoma cell line Caco-2 which expresses PEPT 1 and the SHR rat kidney cell line SKPT which expresses PEPT 2 were used. The uptake of the diPeptide Gly-Sar mediated by PEPT 1 or PEPT 2 in these cells was inhibited significantly by the anionic cephalosporins, with the following order of potency: ceftibuten > cefixime > cefdinir. The inhibition was competitive in nature. Even though the order of potency was the same for PEPT 1 and PEPT 2, PEPT 1 exhibited much lesser sensitivity to inhibition than PEPT 2. In the second approach, the cloned human PEPT 1 and PEPT 2 were functionally expressed in HeLa cells following which the cells were used to study the interaction of anionic cephalosporins with PEPT 1 and PEPT 2. Again, Gly-Sar uptake mediated by the human PEPT 1 and PEPT 2 in HeLa cells was found to be inhibited by the anionic cephalosporins with the same order potency as in Caco-2 and SKPT cells. In the third approach, brush border membrane vesicles isolated from rat kidneys were employed. In this approach also it was found that PEPT 2-mediated Gly-Sar uptake was inhibited by cefixime and ceftibuten. In the fourth approach, the human PEPT 1 was expressed in Xenopus laevis oocytes and PEPT 1-mediated transport of ceftibuten was investigated directly by electrophysiological methods. Ceftibuten evoked inward currents in PEPT 1-expressing oocytes but not in water-injected oocytes, showing that the transport of the anionic cephalosporin via PEPT 1 is associated with transfer of positive charge. The ceftibuten-evoked currents were saturable with respect to ceftibuten concentration and were markedly dependent on membrane potential. It is concluded that anionic cephalosporins interact with the Peptide transporters expressed in the intestine (PEPT 1) as well as in the kidney (PEPT 2).

  • differential recognition of β lactam antibiotics by intestinal and Renal Peptide transporters pept 1 and pept 2
    Journal of Biological Chemistry, 1995
    Co-Authors: Malliga E Ganapathy, Matthias Brandsch, Vadivel Ganapathy, Puttur D Prasad, Frederick H Leibach
    Abstract:

    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.

David E Smith - One of the best experts on this subject based on the ideXlab platform.

  • mechanism of intestinal absorption and Renal reabsorption of an orally active ace inhibitor uptake and transport of fosinopril in cell cultures
    Drug Metabolism and Disposition, 2001
    Co-Authors: Hong Shen, Ulrich Hopfer, David E Smith
    Abstract:

    The objective of this study was to delineate the transepithelial transport mechanisms of fosinopril in cultured cell lines expressing the intestinal and Renal Peptide transporters. Lineweaver-Burk, Dixon, and dose-response analyses revealed that GlySar uptake was competitively inhibited by fosinopril in both Caco-2 ( K i, 35.5 μM) and SKPT cells ( K i, 29.6 μM). Intracellular accumulations of fosinopril were 3 to 4 times higher from apical versus basolateral surfaces of the membrane, as was the apical-to-basal flux of the drug. The apical Peptide transporter had a significantly greater affinity for fosinopril than did the basolateral Peptide transporter in Caco-2 cells ( K m, 154 versus 458 μM, respectively; p < 0.001) and SKPT cells ( K m, 22 versus 104 μM, respectively; p < 0.001). Moreover, fosinopril uptake by the basolateral Peptide transporter was less sensitive to changes in medium pH than the apical Peptide transporter in both cell lines. Although Caco-2 cells are known to express PEPT1 protein (and not PEPT2), our immunoblot analyses provide definitive evidence that SKPT cells express PEPT2 protein (and not PEPT1). Taken as a whole, our findings demonstrate that fosinopril is transported intact by PEPT2 and PEPT1, with high-affinity and by a proton-coupled, saturable process. Our results also suggest that distinct Peptide transporters exist at the basolateral and apical membranes and that they play an important role in modulating the intestinal absorption and Renal reabsorption of Peptides and Peptide-like drugs.

  • mechanism of intestinal absorption and Renal reabsorption of an orally active ace inhibitor uptake and transport of fosinopril in cell cultures
    Drug Metabolism and Disposition, 2001
    Co-Authors: Hong Shen, Ulrich Hopfer, David E Smith
    Abstract:

    The objective of this study was to delineate the transepithelial transport mechanisms of fosinopril in cultured cell lines expressing the intestinal and Renal Peptide transporters. Lineweaver-Burk, Dixon, and dose-response analyses revealed that GlySar uptake was competitively inhibited by fosinopril in both Caco-2 ( K i, 35.5 μM) and SKPT cells ( K i, 29.6 μM). Intracellular accumulations of fosinopril were 3 to 4 times higher from apical versus basolateral surfaces of the membrane, as was the apical-to-basal flux of the drug. The apical Peptide transporter had a significantly greater affinity for fosinopril than did the basolateral Peptide transporter in Caco-2 cells ( K m, 154 versus 458 μM, respectively; p < 0.001) and SKPT cells ( K m, 22 versus 104 μM, respectively; p < 0.001). Moreover, fosinopril uptake by the basolateral Peptide transporter was less sensitive to changes in medium pH than the apical Peptide transporter in both cell lines. Although Caco-2 cells are known to express PEPT1 protein (and not PEPT2), our immunoblot analyses provide definitive evidence that SKPT cells express PEPT2 protein (and not PEPT1). Taken as a whole, our findings demonstrate that fosinopril is transported intact by PEPT2 and PEPT1, with high-affinity and by a proton-coupled, saturable process. Our results also suggest that distinct Peptide transporters exist at the basolateral and apical membranes and that they play an important role in modulating the intestinal absorption and Renal reabsorption of Peptides and Peptide-like drugs.

  • noncompetitive inhibition of glycylsarcosine transport by quinapril in rabbit Renal brush border membrane vesicles effect on high affinity Peptide transporter
    Journal of Pharmacology and Experimental Therapeutics, 1998
    Co-Authors: Wiyada Akarawut, Chunjung Lin, David E Smith
    Abstract:

    Angiotensin converting enzyme (ACE) inhibitors are important therapeutic agents for treating patients with hypertension and cardiovascular diseases. Although most ACE inhibitors are cleared by the kidney via glomerular filtration and tubular secretion, little is known about their reabsorption potential. In particular, it is believed that while certain ACE inhibitors are transported by the intestinal Peptide transporter (PepT1), these same compounds do not interact with the Renal Peptide transporter (PepT2). In the present study, we examined the interaction of quinapril with the high-affinity Peptide transporter, PepT2. Studies were performed in rabbit Renal brush border membrane vesicles in which the uptake of [14C]glycylsarcosine (GlySar), at low substrate concentrations, was examined in the absence and presence of quinapril (and other ACE inhibitors). We found that quinapril was capable of cis-inhibiting the uptake of GlySar and in a concentration-dependent manner. While the Ki for quinapril ( approximately 1 mM) was several-fold higher than the Km for GlySar ( approximately 160 microM), the interaction was unique in that inhibition of PepT2 was of a noncompetitive type. Overall, the data suggest that quinapril is a low-affinity inhibitor of the Renal Peptide transporter and that it binds to a site distinct from that of the GlySar binding site.

Malliga E Ganapathy - One of the best experts on this subject based on the ideXlab platform.

  • transport of valganciclovir a ganciclovir prodrug via Peptide transporters pept1 and pept2
    Journal of Pharmaceutical Sciences, 2000
    Co-Authors: Mitsuru Sugawara, Frederick H Leibach, Vadivel Ganapathy, Wei Huang, Youjun Fei, Malliga E Ganapathy
    Abstract:

    In clinical trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal Peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different experimental systems. We also studied the interaction of these two compounds with the Renal Peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with K(i) values (inhibition constant) of 1.68+/-0.30 and 0.043+/- 0.005 mM, respectively. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiological approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal Peptide transporter PEPT1. This prodrug is also recognized by the Renal Peptide transporter PEPT2 with high affinity.

  • valacyclovir a substrate for the intestinal and Renal Peptide transporters pept1 and pept2
    Biochemical and Biophysical Research Communications, 1998
    Co-Authors: Malliga E Ganapathy, Vadivel Ganapathy, Wei Huang, Hong Wang, Frederick H Leibach
    Abstract:

    Valacyclovir is a prodrug of the antiviral agent acyclovir and it does not contain a Peptide bond in its structure. We studied the interaction of valacyclovir with the Peptide transporters in the human intestinal cell line Caco-2 and the rat kidney proximal tubular cell line SKPT which differentially express Peptide transporters PEPT1 and PEPT2. The results of the studies done with these cell lines were confirmed with the cloned Peptide transporters human PEPT1 and rat PEPT2, expressed heterologously in HeLa cells. The activity of the Peptide transporters was assessed by measuring the uptake of radiolabeled glycylsarcosine in the presence of a H+ gradient. Valacyclovir inhibited the uptake of glycylsarcosine with an inhibition constant (Ki) of 0.49 +/- 0.04 mM in Caco-2 cells and 0.17 +/- 0.01 mM in SKPT cells. In both cell types, the inhibition was competitive. Acyclovir, in contrast to valacyclovir, did not interact with the Peptide transporters. Similar results were obtained with heterologously expressed human PEPT1 and rat PEPT2. Valacyclovir inhibited the hPEPT1-mediated glycylsarcosine transport competitively with a Ki value of 0.74 +/- 0.14 mM. The rPEPT2-mediated transport of glycylsarcosine was also inhibited by valacyclovir competitively and the Ki value for the process was 0.39 +/- 0.03 mM. Acyclovir did not interact with either of these cloned Peptide transporters. We conclude that valacyclovir is a substrate for the Peptide transporters PEPT1 and PEPT2 and that a Peptide bond is not a prerequisite for recognition as a substrate by the Peptide transporters.

  • interaction of anionic cephalosporins with the intestinal and Renal Peptide transporters pept 1 and pept 2
    Biochimica et Biophysica Acta, 1997
    Co-Authors: Malliga E Ganapathy, Vadivel Ganapathy, Puttur D Prasad, Bryan Mackenzie, Frederick H Leibach
    Abstract:

    The present study was undertaken to investigate the interaction of anionic cephalosporins (cefixime, ceftibuten, and cefdinir) with the Renal Peptide transporter (PEPT 2) and the intestinal Peptide transporter (PEPT 1) using four different experimental model systems. In the first approach, the human colon carcinoma cell line Caco-2 which expresses PEPT 1 and the SHR rat kidney cell line SKPT which expresses PEPT 2 were used. The uptake of the diPeptide Gly-Sar mediated by PEPT 1 or PEPT 2 in these cells was inhibited significantly by the anionic cephalosporins, with the following order of potency: ceftibuten > cefixime > cefdinir. The inhibition was competitive in nature. Even though the order of potency was the same for PEPT 1 and PEPT 2, PEPT 1 exhibited much lesser sensitivity to inhibition than PEPT 2. In the second approach, the cloned human PEPT 1 and PEPT 2 were functionally expressed in HeLa cells following which the cells were used to study the interaction of anionic cephalosporins with PEPT 1 and PEPT 2. Again, Gly-Sar uptake mediated by the human PEPT 1 and PEPT 2 in HeLa cells was found to be inhibited by the anionic cephalosporins with the same order potency as in Caco-2 and SKPT cells. In the third approach, brush border membrane vesicles isolated from rat kidneys were employed. In this approach also it was found that PEPT 2-mediated Gly-Sar uptake was inhibited by cefixime and ceftibuten. In the fourth approach, the human PEPT 1 was expressed in Xenopus laevis oocytes and PEPT 1-mediated transport of ceftibuten was investigated directly by electrophysiological methods. Ceftibuten evoked inward currents in PEPT 1-expressing oocytes but not in water-injected oocytes, showing that the transport of the anionic cephalosporin via PEPT 1 is associated with transfer of positive charge. The ceftibuten-evoked currents were saturable with respect to ceftibuten concentration and were markedly dependent on membrane potential. It is concluded that anionic cephalosporins interact with the Peptide transporters expressed in the intestine (PEPT 1) as well as in the kidney (PEPT 2).

  • differential recognition of β lactam antibiotics by intestinal and Renal Peptide transporters pept 1 and pept 2
    Journal of Biological Chemistry, 1995
    Co-Authors: Malliga E Ganapathy, Matthias Brandsch, Vadivel Ganapathy, Puttur D Prasad, Frederick H Leibach
    Abstract:

    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.

Vadivel Ganapathy - One of the best experts on this subject based on the ideXlab platform.

  • transport of valganciclovir a ganciclovir prodrug via Peptide transporters pept1 and pept2
    Journal of Pharmaceutical Sciences, 2000
    Co-Authors: Mitsuru Sugawara, Frederick H Leibach, Vadivel Ganapathy, Wei Huang, Youjun Fei, Malliga E Ganapathy
    Abstract:

    In clinical trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal Peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different experimental systems. We also studied the interaction of these two compounds with the Renal Peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with K(i) values (inhibition constant) of 1.68+/-0.30 and 0.043+/- 0.005 mM, respectively. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiological approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal Peptide transporter PEPT1. This prodrug is also recognized by the Renal Peptide transporter PEPT2 with high affinity.

  • valacyclovir a substrate for the intestinal and Renal Peptide transporters pept1 and pept2
    Biochemical and Biophysical Research Communications, 1998
    Co-Authors: Malliga E Ganapathy, Vadivel Ganapathy, Wei Huang, Hong Wang, Frederick H Leibach
    Abstract:

    Valacyclovir is a prodrug of the antiviral agent acyclovir and it does not contain a Peptide bond in its structure. We studied the interaction of valacyclovir with the Peptide transporters in the human intestinal cell line Caco-2 and the rat kidney proximal tubular cell line SKPT which differentially express Peptide transporters PEPT1 and PEPT2. The results of the studies done with these cell lines were confirmed with the cloned Peptide transporters human PEPT1 and rat PEPT2, expressed heterologously in HeLa cells. The activity of the Peptide transporters was assessed by measuring the uptake of radiolabeled glycylsarcosine in the presence of a H+ gradient. Valacyclovir inhibited the uptake of glycylsarcosine with an inhibition constant (Ki) of 0.49 +/- 0.04 mM in Caco-2 cells and 0.17 +/- 0.01 mM in SKPT cells. In both cell types, the inhibition was competitive. Acyclovir, in contrast to valacyclovir, did not interact with the Peptide transporters. Similar results were obtained with heterologously expressed human PEPT1 and rat PEPT2. Valacyclovir inhibited the hPEPT1-mediated glycylsarcosine transport competitively with a Ki value of 0.74 +/- 0.14 mM. The rPEPT2-mediated transport of glycylsarcosine was also inhibited by valacyclovir competitively and the Ki value for the process was 0.39 +/- 0.03 mM. Acyclovir did not interact with either of these cloned Peptide transporters. We conclude that valacyclovir is a substrate for the Peptide transporters PEPT1 and PEPT2 and that a Peptide bond is not a prerequisite for recognition as a substrate by the Peptide transporters.

  • interaction of anionic cephalosporins with the intestinal and Renal Peptide transporters pept 1 and pept 2
    Biochimica et Biophysica Acta, 1997
    Co-Authors: Malliga E Ganapathy, Vadivel Ganapathy, Puttur D Prasad, Bryan Mackenzie, Frederick H Leibach
    Abstract:

    The present study was undertaken to investigate the interaction of anionic cephalosporins (cefixime, ceftibuten, and cefdinir) with the Renal Peptide transporter (PEPT 2) and the intestinal Peptide transporter (PEPT 1) using four different experimental model systems. In the first approach, the human colon carcinoma cell line Caco-2 which expresses PEPT 1 and the SHR rat kidney cell line SKPT which expresses PEPT 2 were used. The uptake of the diPeptide Gly-Sar mediated by PEPT 1 or PEPT 2 in these cells was inhibited significantly by the anionic cephalosporins, with the following order of potency: ceftibuten > cefixime > cefdinir. The inhibition was competitive in nature. Even though the order of potency was the same for PEPT 1 and PEPT 2, PEPT 1 exhibited much lesser sensitivity to inhibition than PEPT 2. In the second approach, the cloned human PEPT 1 and PEPT 2 were functionally expressed in HeLa cells following which the cells were used to study the interaction of anionic cephalosporins with PEPT 1 and PEPT 2. Again, Gly-Sar uptake mediated by the human PEPT 1 and PEPT 2 in HeLa cells was found to be inhibited by the anionic cephalosporins with the same order potency as in Caco-2 and SKPT cells. In the third approach, brush border membrane vesicles isolated from rat kidneys were employed. In this approach also it was found that PEPT 2-mediated Gly-Sar uptake was inhibited by cefixime and ceftibuten. In the fourth approach, the human PEPT 1 was expressed in Xenopus laevis oocytes and PEPT 1-mediated transport of ceftibuten was investigated directly by electrophysiological methods. Ceftibuten evoked inward currents in PEPT 1-expressing oocytes but not in water-injected oocytes, showing that the transport of the anionic cephalosporin via PEPT 1 is associated with transfer of positive charge. The ceftibuten-evoked currents were saturable with respect to ceftibuten concentration and were markedly dependent on membrane potential. It is concluded that anionic cephalosporins interact with the Peptide transporters expressed in the intestine (PEPT 1) as well as in the kidney (PEPT 2).

  • differential recognition of β lactam antibiotics by intestinal and Renal Peptide transporters pept 1 and pept 2
    Journal of Biological Chemistry, 1995
    Co-Authors: Malliga E Ganapathy, Matthias Brandsch, Vadivel Ganapathy, Puttur D Prasad, Frederick H Leibach
    Abstract:

    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.

Birger Brodin - One of the best experts on this subject based on the ideXlab platform.

  • epidermal growth factor decreases pept2 transport capacity and expression in the rat kidney proximal tubule cell line skpt0193 cl 2
    American Journal of Physiology-renal Physiology, 2004
    Co-Authors: Silvina A Bravo, Carsten Uhd Nielsen, Jan Amstrup, Sven Frokjaer, Birger Brodin
    Abstract:

    The Renal Peptide transporter PEPT2 plays an important role in absorption of di- and tripetides in the proximal tubule; however, knowledge of regulation of PEPT2 by growth factors and hormones is limited. In the present study, we examined the effects of epidermal growth factor (EGF) on PEPT2 transport capacity and expression in the rat proximal tubule cell line SKPT0193 cl.2 (SKPT), which expresses rat PEPT2 (rPEPT2) in the apical membrane. Treatment of SKPT cells with EGF during cell culture growth caused a dose-dependent decrease in rPEPT2 transport capacity and expression, as determined by studies of apical uptake of [14C]glycylsarcosine, rPepT2 mRNA levels, and immunostaining of SKPT cells with a rPEPT2-specific antibody. On the contrary, apical uptake of glucose and lysine was increased in EGF-treated cells, indicating that EGF was not acting generally to decrease apical nutrient uptake mechanisms in the proximal tubule cells. Our findings indicate that EGF decreases rPEPT2 expression by lowering transcription of the rat PepT2 gene or by decreasing rat PepT2 mRNA stability. Previous investigators routinely used SKPT cell culture media with a high (10 ng/ml) EGF concentration. Our study suggests that this might be disadvantageous when studying PEPT2-mediated transport phenomena. These findings demonstrate for the first time EGF-mediated regulation of PEPT2 expression in a kidney cell line. The relevance for kidney regulation of Peptide transport activity in physiological and/or pathophysiological situations, where EGF and EGF receptor levels change drastically, remains to be established.