H+-coupled β-alanine uptake across the apical membrane of intestinal epithelial (Caco-2) cell monolayers (when measured at apical pH 6.5) is inhibited by forskolin but not 1,9-dideoxyforskolin suggesting modulation by a cAMP-dependent pathway (Anderson et al. 2001). The purpose of this study was to investigate the potential (patho)physiological effects of the neuropeptide vasoactive intestinal peptide (VIP) on amino acid absorption via the H+-coupled amino acid transporter hPAT1 (Thwaites et al. 1995; Chen et al. 2003).
Caco-2 cells (passage number 103-117) were cultured on permeable filters (Thwaites et al. 2002) and used 13-18 days post-seeding. β-[3H]Alanine (0.5 µCi ml-1, 100 µM) uptake was measured at apical pH 6.5 (basal pH 7.4) for 15-90 min in the presence or absence of Na+, VIP (0-100 nM) or the selective NHE3 (Na+/H+ exchanger 3) inhibitor S1611 (3 µM) (Wiemann et al. 1999).
Basolateral (but not apical) VIP (5 nM) significantly reduced (P < 0.001, ANOVA, Bonferroni post hoc test) apical β-alanine uptake (15 min) from 283 ± 13 (12) to 154 ± 15 pmol cm-2 (11) (means ± S.E.M. (n)). This effect of VIP was concentration dependent being maximal at 5 nM. In the absence of Na+, VIP had no effect (uptake being 102 ± 11 (9) and 125 ± 12 pmol cm-2 (12) in the absence and presence of VIP, respectively, P > 0.05). The VIP-induced inhibition was through a reduction in the capacity for β-alanine uptake without effect on the affinity. In the presence of Na+, apical S1611 reduced β-alanine uptake to a similar level to that observed in the presence of basolateral VIP.
In conclusion, VIP reduced β-alanine uptake in a Na+-dependent manner consistent with the effect being indirect through inhibition of NHE3. The lack of any apparent direct effect on hPAT1 is supported by the observations that: (i) the NHE3 inhibitor S1611 has a similar effect to VIP on β-alanine uptake; (ii) there are no potential PKA phosphorylation sites within the hPAT1 sequence (Chen et al. 2003); (iii) S1611 and VIP have similar indirect inhibitory effects on H+-coupled dipeptide uptake (Thwaites et al. 2002).
This work was supported by the BBSRC (grant 13/D17277) and MRC (grant G9801704). CMHA is supported by a BBSRC Agri-Food Committee Studentship. S1611 was obtained from H.J. Lang, Aventis Pharma Deutschland GmbH, Chemical Research, Frankfurt/Main, Germany.