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Evaluation of human peptide/histidine transporter 1 (hPHT1/SLC15A4) function: transport kinetics utilizing a HPHT1 shRNA stably transfected knockdown in the hCMEC/D3 blood-brain barrier cell line


Pharmacy & Pharmacology International Journal
David J Lindley,1,2 Stephen M Carl,1,3 Stephanie A Mowery,1,4 Wyatt J Roth,1,5 Gregory T Knipp1

Abstract

Purpose: Investigations of the Proton-dependent Oligopeptide Transporter Superfamily have focused mainly on hPepT1 and hPepT2, with little emphasis on the Peptide/Histidine Transporter isoforms (hPHT1 and hPHT2).1 The studies presented here describe the utilization of small-hairpin RNA (shRNA) technology to establish stable hPHT1 knockdown (KD) and missense (Control) in vitro human blood-brain barrier (BBB) immortalized cell lines (hCMEC/D3).
Methods: Comparative regulation of endogenous transporter mRNA expressions in the hCMEC/D3-hPHT1- Knockdown cells versus the hCMEC/D3-control cells were examined utilizing SuperArray’s 84 drug transporter qRT-PCR array. Monolayer integrity was verified using mannitol and urea. Several POT substrates were examined for hPHT1 specificity using bi-directional permeability studies across these cell lines.
Results: The mRNA transcripts of several drug transporter genes were up or down regulated in the hCMEC/D3-hPHT1-Knockdown cells compared to the hCMEC/D3-Control. Apical→Basolateral (A→B) studies demonstrated increased permeability of valacyclovir and benzylpenacillin in the hCMEC/D3-hPHT1-Knockdown cells comparative to the hCMEC/ D3- Control cells. Additionally, several POT substrates including the newly identified hPHT1 substrate, thiamine showed an increase in Basolateral→Apical (B→A) permeability.
Conclusions: These results suggest that hPHT1 may function differently than PepT1 and PepT2, particularly in the BBB. In addition, hPHT1 may play a neuroprotective role, effluxing substrates out of the basement membrane in the BBB.

Keywords

hCMEC/D3, human proton-dependent oligopeptide transporters, PCR, solute carrier transporters, thiamine, peptide/histidine transporter 1, PHT1, blood-brain barrier

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