Vigabatrin transport across the human intestinal epithelial (Caco-2) brush-border membrane is via the H+ -coupled amino-acid transporter hPAT1

Abstract

The aim of this investigation was to determine if the human proton-coupled amino-acid transporter 1 (hPAT1 or SLC36A1) is responsible for the intestinal uptake of the orally-administered antiepileptic agent 4-amino-5-hexanoic acid (vigabatrin). The Caco-2 cell line was used as a model of the human small intestinal epithelium. Competition experiments demonstrate that [3H]GABA uptake across the apical membrane was inhibited by vigabatrin and the GABA analogues trans-4-aminocrotonic acid (TACA) and guvacine, whereas 1-(aminomethyl)cyclohexaneacetic acid (gabapentin) had no affect. Experiments with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded Caco-2 cells demonstrate that apical exposure to vigabatrin and TACA induce comparable levels of intracellular acidification (due to H+/amino-acid symport) to that generated by GABA, suggesting that they are substrates for a H+ -coupled absorptive transporter such as hPAT1. In hPAT1 and mPAT1-expressing Xenopus laevis oocytes [3H]GABA uptake was inhibited by vigabatrin, TACA and guvacine, whereas gabapentin failed to inhibit [3H]GABA uptake. In Na+ -free conditions, vigabatrin and TACA evoked similar current responses (due to H+/amino-acid symport) in hPAT1-expressing oocytes under voltage-clamp conditions to that induced by GABA (whereas no current was observed in water-injected oocytes) consistent with the ability of these GABA analogues to inhibit [3H]GABA uptake. This study demonstrates that hPAT1 is the carrier responsible for the uptake of vigabatrin across the brush-border membrane of the small intestine and emphasises the therapeutic potential of hPAT1 as a delivery route for orally administered, clinically significant GABA-related compounds.

Figure 1

The effects of GABA and vigabatrin on amino acid uptake across the apical membrane of Caco-2 cell monolayers. Apical uptake of [³H]β-alanine, [³H]proline or [³H]glycine (all 20 μM) (5 min, apical pH 5.5, basolateral pH 7.4, Na⁺-free solutions) in Caco-2 cell monolayers was measured in the absence (open columns) or presence of unlabelled GABA (filled columns) or vigabatrin (hatched columns) (both 10 mM). Data are mean±s.e.m. (n=11): ^**P<0.01 versus control.

Figure 2

The effects of GABA-related compounds on [³H]GABA uptake across the apical membrane of Caco-2 cell monolayers. Apical [³H]GABA (100 μM) uptake (15 min, apical pH 5.5, basolateral pH 7.4, Na⁺-free solutions) in Caco-2 cell monolayers was measured in the presence or absence (control) of unlabelled GABA, vigabatrin, TACA, guvacine or gabapentin (all 10 mM). Data are mean±s.e.m (n=6–12): ^***P<0.001 or ^**P<0.01 both versus control.

Figure 3

Concentration-dependent inhibition of [³H]GABA uptake across the apical membrane of Caco-2 cells by GABA and related compounds. Apical [³H]GABA (20 μM) uptake (15 min, apical pH 5.5, basolateral pH 7.4, Na⁺-free solutions) in Caco-2 cell monolayers was measured in the absence and presence (all 0–10 mM) of unlabelled GABA (filled squares), vigabatrin (filled circles) or TACA (open circles). Data are the mean±s.e.m. (n=17–35). Non carrier-mediated uptake, measured in the presence of excess β-alanine (30 mM), was subtracted from total uptake to determine carrier-mediated uptake.

Figure 4

Intracellular pH measurements in BCECF-loaded Caco-2 cell monolayers. (a) The apical surface of the BCECF-loaded Caco-2 cell monolayers were superfused with a Na⁺-containing pH 7.4 solution (hatched bar). At the indicated point on the diagram the solution was replaced with Na⁺-free pH 5.5 solution (open bar) and subsequently either GABA or vigabatrin (both 10 mM; filled bars) were added to the apical superfusate for 200 s. Replacement of the apical superfusate with a pH 7.4 Na⁺-containing buffer (hatched bar) allows pH_i to recover back towards baseline. The basolateral superfusate was pH 7.4 (Na⁺-free) throughout. A single representative trace of eight. (b) The basolateral surface of the BCECF-loaded Caco-2 cell monolayers were superfused with a Na⁺-containing pH 7.4 solution (hatched bar). At the indicated point on the diagram the solution was replaced with Na⁺-free pH 5.5 solution (open bar) and subsequently either GABA or vigabatrin (both 10 mM; filled bars) were added to the basolateral superfusate for 200 s. Replacement of the basolateral superfusate with a pH 7.4 Na⁺-containing buffer (hatched bar) allows pH_i to recover back towards baseline. The apical superfusate was pH 7.4 (Na⁺-free) throughout. A single representative trace of seven. (c) The effects on pH_i of sequential addition of pH 5.5 (Na⁺-free) alone (open bar) or GABA, vigabatrin and TACA (all 10 mM; filled bars) in a Na⁺-free pH 5.5 buffer. Basolateral pH 7.4, Na⁺-free under all conditions. Where indicated on the figure a Na⁺-containing pH 7.4 solution was superfused across the apical surface to allow the cells to recover (hatched bars). A single trace representative of six.

Figure 5

The effects of GABA and related compounds on amino acid uptake in PAT1-expressing oocytes. (a) [³H]GABA (20 μM) uptake (pH 5.5, Na⁺-free) via hPAT1 was measured in the absence (control) or presence of unlabelled GABA, vigabatrin, TACA, guvacine or gabapentin (all 10 mM). Data are expressed as a percentage control ([³H]GABA uptake in the absence of unlabelled compounds) after subtraction of uptake in water-injected oocytes under identical experimental conditions. Data are mean±s.e.m. (n=18–20): ^**P<0.01 versus control. (b) [³H]GABA, [³H]β-alanine, [³H]glycine, [³H]proline (all 20 μM) uptake (pH 5.5, Na⁺-free) via mPAT1 was measured in the absence (open columns) or presence of GABA (filled columns) or vigabatrin (hatched columns) (both 10 mM). Data are expressed as mean±s.e.m. (n=10) following subtraction of uptake into water-injected oocytes under identical experimental conditions: ^**P<0.01 versus control.

Figure 6

Current changes induced by GABA and related compounds in hPAT1-expressing oocytes. Single representative trace (of three) showing current changes in hPAT1 and water-injected oocytes (insert) following exposure (as indicated by bars) to GABA, vigabatrin, TACA or guvacine (all 10 mM, pH 5.5, Na⁺-free).