Indirect regulation of the intestinal H+-coupled amino acid transporter hPAT1 (SLC36A1).
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.
Anderson
Catriona M H
CM
Faculty of Medical Sciences, Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom.
Thwaites
David T
DT
D000143:Acids / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D026905:Amino Acid Transport Systems / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D020536:Enzyme Activators / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007413:Intestinal Mucosa / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*; D027981:Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014018:Tissue Distribution; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology; D015091:beta-Alanine / Q000037:antagonists & inhibitors
D000143:Acids; D026905:Amino Acid Transport Systems; D020536:Enzyme Activators; D010750:Phosphoproteins; C475905:SLC36A1 protein, human; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D027981:Symporters; C093376:sodium-hydrogen exchanger regulatory factor; D015091:beta-Alanine; D005576:Colforsin; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases