title: The functional evaluation of human peptide/histidine transporter 1 (hPHT1) in transiently transfected COS-7 cells.
abstract: Recently, the expression of the human peptide/histidine transporter (hPHT1, SLC15A4) mRNA was observed in the GI tract and in Caco-2 cells, suggesting that it may participate in the intestinal absorption of peptide-based agents. This study aims to elucidate the: (i) protein expression pattern of hPHT1 (SLC15A4) in human small intestine; (ii) cloning of the hPHT1 full-length sequence; (iii) functional characterization of hPHT1 in transiently transfected COS-7 cells. The expression of hPHT1 was measured using Western blot and immunohistochemical analysis. The hPHT1 full-sequence was amplified from BeWo cells, inserted into the pcDNA3.1-V5/His TOPO plasmid and transiently transfected into COS-7 cells to investigate the uptake kinetics of [3H]histidine and [3H]carnosine. Time, pH and sodium-dependent uptake studies were performed in mock (empty vector) and hPHT1-COS-7 cells. Results demonstrated hPHT1 protein expression in different intestinal regions. Histidine and carnosine uptake was linear in hPHT1-COS-7 cells over 15 min and was found to be pH-dependent. These substrates and valacyclovir showed significantly higher uptake at pH 5.0 in the hPHT1 transients when contrasted to the mock COS-7 cells, whereas glycylsarcosine uptake was significantly lower and unaffected by pH. Other di- and tripeptides also showed affinity for hPHT1. This study presents the initial functional characterization, the protein expression of the hPHT1 transporter and provides insight into a potentially different route for increasing peptide and peptide-based drug transport.
authors.affiliation: Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854-8022, USA.
authors.forename: Rajinder K
authors.initials: RK
authors.lastname: Bhardwaj
authors.forename: Dea
authors.initials: D
authors.lastname: Herrera-Ruiz
authors.forename: Nesreen
authors.initials: N
authors.lastname: Eltoukhy
authors.forename: Maha
authors.initials: M
authors.lastname: Saad
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Knipp
mesh_terms: D000212:Acyclovir / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002336:Carnosine / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007421:Intestine, Small / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection; D000077483:Valacyclovir; D014633:Valine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C491359:SLC15A4 protein, human; D006639:Histidine; D002336:Carnosine; D014633:Valine; D000077483:Valacyclovir; D000212:Acyclovir