Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, C130

Oral Communications

Transport of the photodynamic therapy agent 5-aminolevulinic acid by the amino acid transporter PAT1 (SLC36A1) and the dipeptide transporter PepT1 (SLC15A1).

C. Anderson1, M. Jevons1, T. Muthusamy2, S. Woods1, N. J. Conlon1, V. Ganapathy2, D. T. Thwaites1

1. Epithelial Research Group, Institute for Cell & Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom. 2. Dept. of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia, USA.

5-Aminolevulinic acid (ALA) is a pro-drug used in photodynamic therapy, fluorescent diagnosis and fluorescent-guided resection which produces selective accumulation of protoporphyrin IX in tumour tissue (1). Relatively high oral doses of ALA are used to obtain protoporphyrin IX accumulation in colonic tumours as there is also accumulation in normal gastrointestinal mucosa. Due to the structural similarity of ALA and GABA, a substrate for the H+-coupled amino acid transporter PAT1 (SLC36A1), we investigated the role of PAT1 in luminal ALA uptake. [3H]ALA and [3H]amino acid uptake (10-100μM, 0.5-5μ, Na+ free, pH 5.5 buffer) were measured across the apical membrane of human intestinal Caco-2 cell monolayers (5min, 37°C) grown on permeable filters and in PAT1- or PepT1-expressing X. laevis oocytes (40min, 22°C), as described previously (2). Data are mean ± SEM (n) with ANOVA plus Tukey post-test. In Caco-2 cell monolayers, ALA (10mM) inhibited apical uptake of several PAT1 substrates (e.g. GABA) but not that of other amino acids (e.g. methionine). In PAT1-expressing oocytes, [3H]ALA uptake was significantly greater than that in water-injected oocytes at pH 5.5 (p<0.001) and pH 6.5 (p<0.01) but not at pH 7.4 (p>0.05). PAT1-mediated [3H]ALA uptake was reduced (p<0.001) by 91.6 ± 2.7% (n=20) and 96.4 ± 1.7% (n=20) by GABA and 5-hydroxy-tryptophan [OH-Trp, a PAT1 inhibitor (3)], respectively (both 20mM). ALA is known to be a PepT1 substrate (4). In PepT1-expressing oocytes, [3H]ALA uptake was inhibited by the PepT1 inhibitor 4-aminomethylbenzoic acid [AMBA, 30mM (5)] by 81.8 ± 3.9% (n=20). Rheogenic transport was measured by two-electrode voltage-clamp in PAT1- or PepT1-expressing oocytes (-60mV, Na+ free, pH 5.5). ALA induced current consistent with H+/ALA symport with a Km of 1.6 ± 0.9 mM for PepT1 (n=4) and 10.4 ± 5.6 mM for PAT1 (n=4). In Caco-2 cells, OH-Trp and AMBA significantly (p<0.001) inhibited apical ALA uptake, in an additive manner. The relative expression of PAT1 and PepT1 will determine ALA uptake in normal mucosa and gastrointestinal tumours and, in turn, influence both ALA bioavailability and tumour-specific accumulation of protoporphyrin IX.

Where applicable, experiments conform with Society ethical requirements