T1R taste receptors are present throughout the gastrointestinal tract. Glucose absorption comprises active absorption via SGLT1 and facilitated absorption via GLUT2 in the apical membrane. Trafficking of apical GLUT2 is rapidly up-regulated by glucose and artificial sweeteners, which act through T1R2 + T1R3/α-gustducin to activate PLC β2 and PKC βII. We therefore investigated whether non-sugar nutrients are regulated by taste receptors using perfused rat jejunum in vivo. Under different conditions, we observed a Ca 2+ -dependent reciprocal relationship between the H + /oligopeptide transporter PepT1 and apical GLUT2, reflecting the fact that trafficking of PepT1 and GLUT2 to the apical membrane is inhibited and activated by PKC βII, respectively. Addition of l-glutamate or sucralose to a perfusate containing low glucose (20 mm) each activated PKC βII and decreased apical PepT1 levels and absorption of the hydrolysis-resistant dipeptide l-Phe( S)-l-Ala (1 mm), while increasing apical GLUT2 and glucose absorption within minutes. Switching perfusion from mannitol to glucose (75 mm) exerted similar effects. l-Glutamate induced rapid GPCR internalization of T1R1, T1R3 and transducin, whereas sucralose internalized T1R2, T1R3 and α-gustducin. We conclude that l-glutamate acts via amino acid and glucose via sweet taste receptors to coordinate regulation of PepT1 and apical GLUT2 reciprocally through a common enterocytic pool of PKC βII. These data suggest the existence of a wider Ca 2+ and taste receptor-coordinated transport network incorporating other nutrients and/or other stimuli capable of activating PKC βII and additional transporters, such as the aspartate/glutamate transporter, EAAC1, whose level was doubled by l-glutamate. The network may control energy supply.

Glucose absorption in rat jejunum involves Ca2+ -and PKC βII-dependent insertion of GLUT2 into the apical membrane. Ca 2+ -induced rearrangement of the enterocyte cytoskeleton is thought to enhance paracellular flow. We have therefore investigated the relationships between myosin II regulatory light chain phosphorylation (RLC 20 ), absorption of glucose, water and calcium, and mannitol clearance. ML-7, an inhibitor of myosin light chain kinase, diminished the phloretin-sensitive apical GLUT2 but not the phloretin-insensitive SGLT1 component of glucose absorption in rat jejunum perfused with 75 mM glucose. Western blotting and immunocytochemistry revealed marked decreases in RLC 20 phosphorylation in the terminal web and in the levels of apical GLUT2 and PKC βII, but not SGLT1. Perfusion with phloridzin or 75 mM mannitol, removal of luminal Ca 2+ , or inhibition of unidirectional 45 Ca 2+ absorption by nifedipine exerted similar effects. ML-7 had no effect on the absorption of 10 mM Ca 2+ , nor clearance of [14 C]-mannitol, which was less than 0.7% of the rate of glucose absorption. Water absorption did not correlate with 45 Ca 2+ absorption or mannitol clearance. We conclude that the Ca 2+ necessary for contraction of myosin II in the terminal web enters via an L-type channel, most likely Ca v 1.3, and is dependent on SGLT1. Moreover, terminal web RLC 20 phosphorylation is necessary for apical GLUT2 insertion. The data confirm that glucose absorption by paracellular flow is negligible, and show further that paracellular flow makes no more than a minimal contribution to jejunal Ca 2+ absorption at luminal concentrations prevailing after a meal.

4-Aminophenylacetic acid (4-APAA), a peptide mimic lacking a peptide bond, has been shown to interact with a proton-coupled oligopeptide transporter using a number of different experimental approaches. In addition to inhibiting transport of labeled peptides, these studies show that 4-APAA is itself translocated.4-APAA transport across the rat intact intestine was stimulated 18-fold by luminal acidification (to pH 6.8) as determined by high performance liquid chromatography (HPLC); in enterocytes isolated from mouse small intestine the intracellular pH was reduced on application of 4-APAA, as shown fluorimetrically with the pH indicator carboxy-SNARF; 4-APAA trans-stimulated radiolabeled peptide transport in brush-border membrane vesicles isolated from rat renal cortex; and in Xenopus oocytes expressing PepT1, 4-APAA produced trans-stimulation of radiolabeled peptide efflux, and as determined by HPLC, was a substrate for translocation by this transporter.These results with 4-APAA show for the first time that the presence of a peptide bond is not a requirement for rapid translocation through the proton-linked oligopeptide transporter (PepT1). Further investigation will be needed to determine the minimal structural requirements for a molecule to be a substrate for this transporter.The rapid uptake of intact small peptides across the brushborder membrane of the small intestinal epithelium is the major route for absorption of dietary protein ␣-amino nitrogen (1). Hitherto, it has been thought that a number of chemical features, for example free amino and carboxyl termini, are essential in contributing to substrate interaction with, and translocation through, the intestinal peptide transporter.These features include the presence of a peptide bond within the substrate molecules. Indeed a major review (1) states that "it is the presence of peptide bonds which make di-and tripeptide acceptable to the peptide transport systems." Although previous work (e.g. Ref.2) has shown that molecules lacking this feature can inhibit transport of peptides (presumably by substrate binding), we describe here for the first time rapid transport of a small totally non-peptidic substrate through the intestinal peptide transporter. The substrate, 4-aminophenylacetic acid (4-APAA), 1 was selected on the basis of its chemical structure, it being a potential mimic of a dipeptide (D-Phe-LAla) (Fig. 1) which previously we have shown to be an excellent substrate for epithelial peptide transport (3, 4). EXPERIMENTAL PROCEDURESRat renal brush-border membrane vesicles were prepared as described previously (5), and initial rates of labeled peptide transport (influx, efflux) were determined by rapid filtration (4, 6). Rat intestinal loops in vitro and vascularly perfused small intestine in situ were used to measure transepithelial fluxes in the intact small intestine as described previously (3, 7). Luminal pH was changed using a previously published protocol (8). Isolated murine enterocytes were prepared by enzymatic digestion using haluronidase, and i...

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Four hydrolysis-resistant dipeptides (D-phenylalanyl-L-alanine, D-phenylalanyl-L-glutamine, D-phenylalanyl-L-glutamate and D-phenylalanyl-L-lysine) were synthesized to investigate the effects of net charge on transmural dipeptide transport by isolated jejunal loops of rat small intestine. At a luminal pH of 7.4 and a concentration of 1 mM the two dipeptides with a net charge of -1 and +1 were transported at substantially slower rates (18 +/- 1.3 and 8.4 +/- 1.3 nmol min(-1)(g dry wt.)(-1), respectively) than neutral D-phenylalanyl-L-alanine and D-phenylalanyl-L-glutamine (87 +/- 0.2 and 197 +/- 14 nmol min(-1)(g dry wt.)(-1), respectively). We investigated the effects of luminal pH on dipeptide transport by varying the NaHCO3 content of Krebs Ringer perfusate equilibrated with 95% 02/5% CO2. The pH changes did not affect water transport, but serosal glucose appearance increased significantly at pH 6.8. Transmural transport of D-phenylalanyl-L-alanine and D-phenylalanyl-L-glutamine at pH 6.8 was stimulated (P < 0.01) by 61% and 49%, respectively, whereas the lower pH increased the rate for negatively charged D-phenylalanyl-L-glutamate by 306% (P < 0.01) and decreased that for positively charged D-phenylalanyl-L-lysine by 46% (P < 0.05). Increasing luminal pH to 8.0 inhibited D-phenylalanyl-L-alanine transport by 60%, whereas D-phenylalanyl-L-lysine transport was 60% faster.