D‐Cycloserine transport in human intestinal epithelial (Caco‐2) cells: mediation by a H ⁺ ‐coupled amino acid transporter

Abstract

Medical School, Newcastle upon Tyne, NE2 4HH1 The ability of D-cycloserine to act as a substrate for H+/amino acid symport has been tested in epithelial layers of Caco-2 human intestinal cells. 2 In Na+-free media with the apical bathing media held at pH 6.0, D-cycloserine (20 mM) is an effective inhibitor of net transepithelial transport (Jnet) of L-alanine (100 pM) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L-alanine, /3-alanine, L-proline and glycine). In contrast L-valine was ineffective as an inhibitor for H'/amino acid symport. Both inhibition of L-alanine Jne, and its accumulation by D-cycloserine were dose-dependent, maximal inhibition being achieved by 5-10 mM. 3 Both D-cycloserine and known substrates for H+/amino acid symport stimulated an inward short circuit current (ISC) when voltage-clamped monolayers of Caco-2 epithelia, mounted in Ussing chambers, were exposed to apical substrate in Na+-free media, with apical pH held at 6.0. The D-cycloserine dependent increase in ISC was dose-dependent with an apparent Km = 15.8 + 2.0 (mean + s.e.mean) mm, and Vma,, = 373 + 21 nmol cm-2 h-'. 4 D-Cycloserine (20 mM) induced a prompt acidification of Caco-2 cell cytosol when superfused at the apical surface in both Na+ and Na+-free conditions. Cytosolic acidification in response to D-cycloserine was dependent upon superfusate pH, being attenuated at pH 8 and enhanced in acidic media. 5 The increment in ISC with 20 mM D-cycloserine was non-additive with other amino acid substrates for H+/amino acid symport.

H+/solute-induced intracellular acidification leads to selective activation of apical Na+/H+ exchange in human intestinal epithelial cells

“…This amino acid transport is coupled with proton inflow in monolayers of Caco-2 cells that have been loaded with the pH-sensitive fluorescent dye BCECF (BCECF; refs. 7,8). These observations emphasize the potential importance of the H + electrochemical gradient as a driving force of solute absorption across the mammalian intestinal wall.…”

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

“…Although direct measurement of transepithelial transport of vigabatrin is necessary to verify that vigabatrin exits the basolateral membrane of these intestinal epithelial cells, previous investigations with other PAT1 substrates, namely β ‐alanine, L ‐alanine, α ‐methylaminoisobutyric acid, proline, GABA and glycine ( Thwaites et al ., 1993a , 1993b ; 1994 ; 1995a , 1995b , 1995c ; 2000 ), when considered alongside the high oral bioavailability of vigabatrin, suggest that transepithelial transport occurs. Potential efflux systems localised to the basolateral membrane of the small intestine include system L (CD98/LAT2, SLC3A2/SLC7A8) which exchanges neutral amino acids ( Rossier et al ., 1999 ; Bauch et al ., 2003 ), system y + L (CD98/y + LAT1, SLC3A2/SLC7A7), which exchanges neutral amino acids for intracellular cationic amino acids in the presence of sodium ( Kanai et al ., 2000 ; Bauch et al ., 2003 ), or system asc (CD98/asc1, SLC3A2/SLC7A10), which is a Na + ‐independent, high‐affinity neutral amino‐acid exchanger ( Wagner et al ., 2001 ).…”

Indirect regulation of the intestinal H+‐coupled amino acid transporter hPAT1 (SLC36A1)

“…Recent studies of hPAT1 substrate specificity have confirmed these earlier reports which showed that hPAT1 may play a unique role in the small intestine by acting as the only high‐capacity mechanism for uptake of the conditionally essential amino acid taurine and neuroactive D ‐amino acids such as D ‐serine (Thwaites et al, 1995b; Thwaites and Stevens, 1999; Chen et al, 2003). hPAT1 transports potential therapeutic compounds such as D ‐cycloserine and betaine (Thwaites et al, 1995a,b; Boll et al, 2003; Anderson et al, 2004). Recently, it has been identified that PAT1 can also transport short chain fatty acids such as butyrate (Foltz et al, 2004).…”

“…Details of what happens to hPAT1‐mediated amino acid absorption during such disease states has yet to be studied in detail but we would suggest that excessive VIP would exacerbate the generalised mal‐absorption associated with diarrhoea due to downregulation of pro‐absorptive mechanisms involved in both nutrient and salt absorption. Inhibition of hPAT1 by VIP in such situations would result in malabsorption of not only essential amino acids but also a number of therapeutic agents such as D ‐cycloserine (used as an antibiotic) (Thwaites et al, 1995a). This may be particularly significant in infant diarrhoea where the potential for mal‐absorption of dietary taurine [a hPAT1 substrate (Thwaites and Stevens, 1999)] may have prominent effects on development (Sturman, 1993).…”

“…Recently, a H + ‐coupled amino acid transporter, hPAT1, has been identified as being present at the brush border membrane of intestinal Caco‐2 cells and human and rat small intestine (Chen et al, 2003; Anderson et al, 2004). hPAT1 mediates pH‐dependent, Na + ‐independent uptake of essential and semi‐essential amino acids such as glycine, alanine, proline, and taurine (Thwaites et al, 1995b; Thwaites and Stevens, 1999) as well as potential therapeutic compounds such as D ‐cycloserine (Thwaites et al, 1995a) [used as an antibiotic and to treat schizophrenia (Evins et al, 2002)], betaine (Thwaites et al, 1995b; Boll et al, 2003) [used in the treatment of homocystinuria (Smolin et al, 1981)], and analogues of GABA (Thwaites et al, 2000; Anderson et al, 2004) [which can be used as GABA receptor agonists or reuptake inhibitors (Krogsgaard‐Larsen et al, 1987)].…”