Norma Lister

An energy supply network of nutrient absorption coordinated by calcium and T1R taste receptors in rat small intestine

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 Ca2+‐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(ΨS)‐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 Ca2+ 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.

Peptide Mimics as Substrates for the Intestinal Peptide Transporter

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-APAAtrans-stimulated radiolabeled peptide transport in brush-border membrane vesicles isolated from rat renal cortex; and inXenopus oocytes expressing PepT1, 4-APAA producedtrans-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.

Dipeptide transport and hydrolysis in isolated loops of rat small intestine: effects of stereospecificity.

1. Isolated jejunal loops of rat small intestine were perfused by a single pass of bicarbonate Krebs‐Ringer solution containing either D‐ or L‐phenylalanine or one of eight dipeptides formed from D‐ or L‐alanine plus D‐ or L‐phenylalanine. 2. At 0.5 mM L‐phenylalanyl‐L‐alanine increased serosal phenylalanine appearance to forty times the control rate giving a value similar to that found with 0.5 mM free L‐phenylalanine. No serosal dipeptide could be detected. 3. Perfusions with the two mixed dipeptides with N‐terminal D‐amino acids (D‐alanyl‐L‐phenylalanine and D‐phenylalanyl‐L‐alanine) gave rise to the appearance of intact dipeptides in the serosal secretions although there were substantial differences in their rates of absorption and subsequent hydrolysis. 4. L‐Alanyl‐D‐phenylalanine was absorbed from the lumen three to five times as fast as L‐phenylalanyl‐D‐alanine. At 1 mM L‐alanyl‐D‐phenylalanine transferred D‐phenylalanine across the epithelial layer at more than seven times the rate found with the same concentration of the free D‐amino acid. 5. Perfusions with D‐alanyl‐D‐phenylalanine or D‐phenylalanyl‐D‐alanine showed that these two dipeptides are poor substrates for both transport and hydrolysis by the rat small intestine. 6. Analysis of mucosal tissue extracts after perfusion with the two mixed dipeptides with N‐terminal D‐amino acids revealed that both dipeptides were accumulated within the mucosa and suggested that exit across the basolateral membrane was rate limiting for transepithelial dipeptide transport.

Calcium absorption by Cav1.3 induces terminal web myosin II phosphorylation and apical GLUT2 insertion in rat intestine

Glucose absorption in rat jejunum involves Ca2+‐ and PKC βII‐dependent insertion of GLUT2 into the apical membrane. Ca2+‐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 (RLC20), 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 RLC20 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 Ca2+, or inhibition of unidirectional 45Ca2+ absorption by nifedipine exerted similar effects. ML‐7 had no effect on the absorption of 10 mm Ca2+, nor clearance of [14C]‐mannitol, which was less than 0.7% of the rate of glucose absorption. Water absorption did not correlate with 45Ca2+ absorption or mannitol clearance. We conclude that the Ca2+ necessary for contraction of myosin II in the terminal web enters via an L‐type channel, most likely Cav1.3, and is dependent on SGLT1. Moreover, terminal web RLC20 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 Ca2+ absorption at luminal concentrations prevailing after a meal.

The influence of luminal pH on transport of neutral and charged dipeptides by rat small intestine, in vitro

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.

Identification of a candidate membrane protein for the basolateral peptide transporter of rat small intestine.

A candidate protein for the basolateral peptide transporter of rat jejunum is described. Vascular perfusion of the photoaffinity label, [4-azido-D-phe]-L-ala (2.5mM), had no effect on the transepithelial transport of the non-hydrolysable dipeptide D-phe-L-gln (1mM) from the lumen, its mucosal accumulation or wash-out into the vascular perfusate. When the label was perfused luminally, the transepithelial transport of D-phe-L-gln was inhibited by 38% (P<0.001) and accumulation increased by 62% (P<0.05). These data are consistent with those of a basolateral transporter that is strongly asymmetric in its substrate binding and transport properties. Labelling of basolateral membrane vesicles with [4-azido-3,5-3H-D-phe]-L-ala revealed that the majority of label was incorporated into a single protein of M(r)112+/-2 kDa and pI 6.5. MALDI-TOF analysis of tryptic digests of the protein followed by database searches established that this protein was novel with no obvious similarity to PepT1, the apical membrane transporter.

Tripeptide transport in rat lung

Transport of L-alanyl-D-phenylalanyl-L-alanine was investigated with an in situ vascular perfusion preparation of rat lung and brush border membrane vesicles prepared from type II pneumocytes. In the perfused lung 1 mM tripeptide was transported intact from the alveolar lumen to the vascular perfusate at a mean rate of 25.1 +/- 1.29 (3) nmol/min per g dry weight. D-Phenylalanine also appeared in the vascular perfusate at a rate of 21.9 +/- 1.74 (3) nmol/min per g dry weight indicating that 47% of the absorbed tripeptide was split during passage across the epithelial layer. No dipeptide could be detected in the vascular effluent during perfusions with tripeptide. Rapid L-alanyl-D-phenylalanyl-L-alanine uptake occurred with fresh apical membrane vesicles prepared from type II pneumocytes and this was abolished by treatment with 0.1% triton. The related tripeptide, D-alanyl-L-phenylalanyl-D-alanine, was taken up significantly more slowly by the vesicles. D-phenylalanyl-L-alanine and D-phenylalanyl-D-alanine, were also studied with the vascularly perfused preparation; the mixed dipeptide appeared in the vascular perfusate significantly faster than L-alanyl-D-phenylalanyl-L-alanine whereas D-phenylalanyl-D-alanine appeared more slowly and was not hydrolysed.

Dipeptide transport and hydrolysis in rat small intestine, in vitro

A range of natural and mixed D-/L-stereoisomer phenylalanine dipeptides was used to investigate peptide uptake and hydrolysis by isolated rings of rat jejunum. Characterisation of dipeptide hydrolysis by the brush border fraction revealed apparent Km values in the 0.1-1.0 mM range which, except for the charged dipeptides, were significantly higher than those for hydrolysis by the cytosolic fraction. Uptake of L-/L-dipeptides into jejunal rings, which was followed by HPLC, was unaffected by the presence of peptidase inhibitors in the incubation medium although the absorbed peptides were completely hydrolysed in the cytosol; comparison of the effects of excess leucine on dipeptide uptake and on the uptake of the two constituent amino acids were also consistent with absorption of intact dipeptide followed by cytosolic hydrolysis. The uptake of hydrolysis-resistant mixed D-/L-dipeptides was also studied and confirmed that peptide uptake preceded hydrolysis; D-alanyl-L-phenylalanine accumulated within the rings to twice the medium concentration.