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

Abstract

A H(+)-coupled amino acid transporter has been characterised functionally at the brush border membrane of the human intestinal cell line Caco-2. This carrier, hPAT1 (human Proton-coupled Amino acid Transporter 1) or SLC36A1, has been identified recently at the molecular level and hPAT1 protein is localised to the brush border membrane of human small intestine. hPAT1 transports both amino acids (e.g., beta-alanine) and therapeutic agents (e.g., D-cycloserine). In human Caco-2 cells, hPAT1 function (H(+)/amino acid symport) is associated with a decrease in intracellular pH (pH(i)), which selectively activates the Na(+)/H(+) exchanger NHE3, and thus maintains pH(i) and the driving force for hPAT1 function (the H(+) electrochemical gradient). This study provides the first evidence for regulation of hPAT1 function. Activation of the cAMP/protein kinase A pathway in Caco-2 cell monolayers either using pharmacological tools (forskolin, 8-br-cAMP, [(11,22,28)Ala]VIP) or physiological activators (the neuropeptides VIP and PACAP) inhibited hPAT1 function (beta-alanine uptake) at the apical membrane. Under conditions where NHE3 is inactive (the absence of Na(+), apical pH 5.5, the presence of the NHE3 inhibitor S1611) no regulation of beta-alanine uptake is observed. Forskolin and VIP inhibit pH(i) recovery (NHE3 function) from beta-alanine-induced intracellular acidification. Immunocytochemistry localises NHERF1 (NHE3 regulatory factor 1) to the apical portion of Caco-2 cells where it will interact with NHE3 and allow PKA-mediated phosphorylation of NHE3. In conclusion, we have shown that amino acid uptake via hPAT1 is inhibited by activators of the cAMP pathway indirectly through inhibition of NHE3 activity.

Leptin regulates sugar and amino acids transport in the human intestinal cell line Caco‐2

“…As previously mentioned, activity of the apical Na + /H + exchanger maintains the H + electrochemical gradient necessary for the correct functionality of PAT1 (Anderson et al 2004, Seidler et al 2008. Moreover, it has been demonstrated that activation of cAMP/PKA pathway decreases the activity of the Na + /H + exchanger NHE3 (Anderson & Thwaites 2005) and indirectly inhibits PAT1, in line with the fact that PAT1 has no intracellular PKA phosphorylation sites (Chen et al 2004). On the other hand, it is well known that leptin is able to activate PKA (Barrenetxe et al 2004, Than et al 2011 It has been reported in monolayer of Caco-2 cells grown and differentiated on permeable supports that luminal leptin produces, after 30 min incubation, an increase in both uptake of dipeptides by the H + -dependent transporter PEPT1 and translocation of the transporter from the intracellular pool to the plasma membrane (Buyse et al 2001).…”

“…(Fig. 5a) Because amino acid uptake via human PAT1 in Caco-2 cells is inhibited by activators of the cAMP pathway through inhibition of the Na + /H + exchanger 3 (NHE3) activity (Anderson et al 2005) and the mechanism of inhibition of β-Ala uptake by leptin did not seem to involve decrease of PAT1 abundance in BBMV, we decided to investigate whether a PKA-dependent pathway could be implicated in this leptin effect. The uptake of 1 mM β-Ala was measured in Na + medium at pH 6 in the presence of 8 nM leptin in control conditions and after pre-incubation of the cells with 1 µM H-89.…”

Isolation and function of the amino acid transporter PAT1 (slc36a1) from rabbit and discrimination between transport via PAT1 and system IMINO in renal brush-border membrane vesicles

“…Previously we have identified that hPAT1 function in small intestinal epithelia is partially Na ' -dependent due to a requirement for the coexpression and cooperative activity of the Na ' /H ' exchanger NHE3 Anderson et al 2004;Anderson & Thwaites 2005). Whether there is a similar relationship between PAT1 and NHE3 function in the renal proximal tubule is not known but low-affinity proline influx into rat kidney cortex slices also shows partial Na ' -dependence being reduced by 31% in the absence of Na ' (Mohyuddin & Scriver 1970).…”

Dual modes of 5-(N-ethyl-N-isopropyl)amiloride modulation of apical dipeptide uptake in the human small intestinal epithelial cell line Caco-2

“…During the absorptive process, the intestinal epithelial cell must be able to establish and maintain the transmembrane ionic gradients so that optimal absorption is achieved. Thus, the maximal absorptive capacity of the intestinal epithelial brush-border surface for transport of small di/tripeptides and peptide-like drugs via hPepT1 is also dependent upon the coexpression and cooperative functional activity of the Na + /H + exchanger NHE3 so that peptide-cotransported protons are recycled back across the brush-border surface to maintain the driving force for further hPepT1-mediated transport [16,20,21,23,28,29]. Indeed, NHE3 is normally quiescent at resting pH i values but is activated by transport through the H + -coupled di/tripeptide transporter hPepT1 [23].…”

“…Thus inhibition of NHE3 by removal of extracellular Na + indirectly inhibits hPepT1. This requirement for cooperative functional activity with NHE3 is not limited to hPepT1 as, in intact epithelial preparations, NHE3 activity is also required for optimal transport via the H + -coupled amino acid transporter hPAT1 [28,29]. Up-or down-regulation of NHE3 activity by physiological, pathophysiological or pharmacological means will, therefore, also indirectly control the intestinal absorptive capacity for transport via H + -coupled transporters such as hPepT1 and hPAT1.…”