A H+-coupled amino acid transporter has been identified in human intestinal Caco-2 cell monolayers (Thwaites et al. 1993). In general relatively little is known about regulation of intestinal amino acid absorption. The aim of this investigation was to identify whether or not the H+-coupled amino acid carrier is regulated by the protein kinase A (PKA) pathway (activated using forskolin) in a similar fashion to that observed with the H+-coupled di/tripeptide transporter hPepT1 (Thwaites et al. 1999).
Caco-2 cell monolayers (passage number 98-118, 12-18 days post-seeding) were grown on permeable polycarbonate filters (12 mm in diameter). [3H] β-alanine uptake (0.5 µCi ml-1, 100 µM, 15 min, 37 °C) across the apical membrane was determined in modified Krebs-Ringer solution (apical pH 5.0-7.4, basal pH 7.4) containing either 137 mM NaCl or 137 mM choline chloride (Na+-free), essentially as described previously (Thwaites et al. 1993). In parallel experiments, monolayers were loaded with BCECF to allow measurement of pHi recovery after β-alanine-induced acidification.
Forskolin (10 µM) had a dual effect on β-alanine uptakes (mean ± S.E.M. (n)), uptake being reduced at pH 7.4 (150 ± 9 (13) to 104 ± 3 (13) pmol cm-2) and pH 6.5 (808 ± 41 (13) to 512 ± 16 (13) pmol cm-2) but enhanced at pH 5.5 (1654 ± 98 (9) to 2267 ± 81 (9) pmol cm-2) (P < 0.0005 at each pH versus control, Student’s t test). In contrast 1, 9-dideoxyforskolin (10 µM) was without effect. In Na+-free conditions, forskolin was without effect at either pH 6.5 or 7.4. In BCECF-loaded Caco-2 monolayers, the Na+-dependent rate of pHi recovery after acid loading by exposure to 20 mM β-alanine (at pH 5.5) was dependent on apical pH (being reduced at pH 5.5), and was inhibited by forskolin (10 µM) but not 1, 9-dideoxyforskolin (10 µM).
In conclusion, there appear to be two effects of forskolin on β-alanine uptake. Firstly, a Na+-dependent inhibitory effect over the apical pH range 6.5-7.4, which is consistent with inhibition of the apical Na+-H+ exchanger NHE3 (and a reduction in the driving force (the proton electrochemical gradient) for β-alanine uptake). Secondly, forskolin appears to stimulate β-alanine uptake at pH 5.5. Since NHE3 is inactive at pH 5.5 (Orlowski, 1993) this forskolin-induced stimulation of β-alanine uptake could represent a direct effect on transporter capacity at the apical membrane.
This work was supported by the BBSRC (grant number 13/D09145). C.M.H.A. is supported by a BBSRC Agri-Food Committee Studentship.
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