Development of Dipeptide Transport in the Intestinal Mucosa of Rabbits
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Pediatr. Res. 16: 99-103 (1982) Development of Dipeptide Transport in the Intestinal Mucosa of Rabbits STEFAN0 GUANDALINI'32' AND ARMIDO RUBINO Child Health Institute, 2nd School of Medicine, University of Naples, Italy Summary other hand, they can also be hydrolyzed at the brush border membrane, with-subsequent entjof the released amino acids. The uptake of 0.5 mM glycyl-L-proline (Gly-Pro), a substrate We had previously shown (24) that both ways do occur also in which has affinity for the dipeptide specific carrier system and is in the small intestine of the newborn rabbit, the transport of intact not hydrolyzed in the brush border, was studied vitro in jejunum dipeptide being quantitatively approximately as important as the and ileum of rabbits from the 25th day of gestational age into other pathway. We thus moved to investigate, in the developing adulthood. For comparison, uptake of 0.5 mM glycine was studied rabbit, the carrier system for dipeptides, with these major aims: too. Results show that influx of Gly-Pro is present from the (1) provide a biologic characterization of the system as for sub- earliest age studied and show a steep prenatal increase and peaks straie specificity, ~a dependence and kinetic constants; (2) define f at birth, with values, in the newborn jejunum, of 45.2 3.3 pmoles/ its developmental pattern as compared to that of the uptake of a ghr (mean + S.E.). Thereafter, the uptake slowly declines toward free neutral amino acid, as to provide an ontogenetic basis for the the adult level of 3.8 f 0.5 pmoles/ghr. A similar pattern was distinction between the two systems; (3) assess, in the youngster, present in the ileum. On the contrary, free glycine uptake shows the relative importance of dipeptide vs. free amino acid uptake. no statistically significant change during the whole period studied. Therefore, the uptakes of glycyl-proline labeled in the glycine The characteristics of the Gly-Pro influx process have been residue and, for comparison, that of free glycine, have been studied studied in the period of its peak activity (1-to dday-old rabbits) in in vitro in jejunal and ileal segments of fetal, newborn, suckling, both jejunum and ileum. The process is not inhibited by a 40-fold and adult rabbits. excess of glycine, proline, phenylalanine, leucine, lysine, glutamic Glycyl-proline has been chosen as a substrate, because it has acid, while it is strongly inhibited by a 10-fold excess of the affinity for the dipeptide carrier system but is not hydrolyzed in dipeptides leucyl-leucine, alanyl-proline, glycyl-leucine and lysyl- the brush border, its hydrolysis being accomplished entirely intra- leucine. A large excess of Gly-Pro has no inhibitory effect on the cellularly (20, 29). uptake of free glycine. The kinetic constants for Gly-Pro uptake are: Vmax = 126.7 26.4 pmoles/ghr and Kt = 0.98 0.26 mM + + MATERIALS AND METHODS in the jejunum and Vmax = 59.6 k 9.2 and Kt = 0.70 + 0.10 in the ileum. The removal of Na from the incubation and preincubation MEASUREMENT OF UNIDIRECTIONAL INFLUX OF SUBSTRATES medium approximately halves the Vmax in both segments,- leaving- FROM LUMEN INTO THE EPITHELIUM unchangedthe affinity constants. These results indicate that a very efficient dipeptide influx New Zealand White rabbits were used: fetuses (from the 25th process occurs in the newborn rabbit. This process displays many to the 30th day of gestational age), newborns (1 to 6 days old), characteristics in common with that described in the adult: it is suckling and weaned (at the loth, 20th, 30th, 40th, and 50th day not shared by amino acids free in the lumen; has a broad specificity of postnatal age), and adults (older than 3 months). for dipeptides and shows the same pattern of Na dependence. For studies in the fetuses, the pregnant rabbit was killed by However, its maximal velocity is in the newborn much higher than cervical dislocation and the fetuses, quickly removed from the in the adult. uterus, were then killed by decapitation. Therefore, (1) the developmental patterns of the transport sys- The newborns had always been fed by their mothers (as judged tems for Gly-Pro and for free glycine are different; this provides by the presence of milk in the stomach), and were also killed by ontogenetic evidence for the distinction between dipeptide and decapitation. free amino acid carrier systems; and (2) dipeptide uptake plays a The animals beyond 10 days of postnatal life were killed by predominant role in the physiology of the protein absorptive- cervical dislocation. digestive processes of the youngster. In all cases, the intestinal segment under study was quickly excised, opened along- the mesenteric border, and rinsed free of intestinal contents with a Ringer solution. Speculation In the fetuses, studies were done on the proximal and distal Dipeptide uptake is likely to play a major role in the final stages third of the small intestine, heretofore referred to as jejunum and of protein absorption in the youngster. Thus, wherever proteic oral ileum, respectively. In the newborns and thereafter, the small supplementation is indicated in the newborn or young infant, the intestine (distal to the ligament of Treitz) was divided in five administration of free amino acids does not appear to be physio- segments: the first two-fifths referred to as proximal and distal logically appropriate. jejunum, and the last two-fifths referred to as proximal and distal ileum; the middle fifth was discarded. Influx measurements were then carried out in the adult animals The final products of intraluminal digestion of protein are low according to the technique previously described (23). In all the molecular weight peptides and free amino acids (6). A substantial animals of younger age, the same basic technique was followed, body of evidence accumulated in the adult animal has shown that with some appropriate modifications. In these ages, the following di- and tripeptides can be transported into the intestinal cells by procedure was used: the segment under study was preincubated a specific carrier system which is different from those involved in for 30 min in a Ringer solution having, unless otherwise indicated, the transport of free amino acids (reviewed in Ref. 19). On the the following composition: NaC1, 140 mM; KHCOs, 10 mM; 100 GUANDALINI AND RUBINO K2HP04,1.2 mM; KH2P04,0.2 mM; CaC12, 1.2 mM; and MgC12, Statistical evaluation of the data was done by Student's t test 1.2 mM (pH 7.2). Preincubation was carried out at 37OC and with for unpaired or, where appropriate for paired values. All values bubbling 02-CO2 (95 to 5%). are expressed as means f 1 S.E. Pieces (100 to 300 mg) of the intestinal segment were then individually placed in an Erlenmeyer flask containing the test solution. This was a Ringer solution of identical composition, but RESULTS containin the radioactively labeled compound under study (either (Glycyl-' FC) glycyl-proline or 3H-glycine)and inulin (labeled with STUDIES IN THE NEWBORN either % or I4C) as a marker of the extracellular space. The incubation in the test solution described was also carried out at Uptake of (Glycyl-14C) Glycyl-L-proline and of 3H-Glycine by 37°C and with gassing 02-C02 (95 to 5%). After 50 to 60 sec, the Small Intestine of Newborn Rabbit. The uptakes of 0.5 mM Gly- piece was quickly removed, rinsed in cold 0.3 M Mannitol solution, Pro and of 0.5 mM Gly by segments of jejunum-and ileum of blotted on filter paper, placed in 2 ml of 10% thrichloroacetic acid newborn (1 to 6 days old) and for comparison of adult animals (TCA), and weighed. After homogeneization by means of an are reported in Table 1. Gly-Pro is taken up by the small intestine Ultra-Turrax tissue homogenizer, tissues were centrifuged at 5000 of newborns in much larger amount than an equimolar amount of x g for 8 min and aliquots of the supernatants were then added Gly; this is also true, though to a lesser extent, in the jejunum of to Bray's solution and assayed for radioactivity in a liquid scintil- the adult. Furthermore, in both the intestinal segments tested, lation counter. Aliquots (50 to 100 p1) of the media were similarly Gly-Pro uptake is much larger in the newborn than in the adult counted; the total content of 10% TCA in all scintillation vials animal (all these differences being statistically highly significant). was kept constant, so as to minimize quenching variations among Finally, a different distribution pattern for Gly-Pro and Gly different samples. uptake is seen along the small intestine of the newborn: sites of Calculations were done accordingly to the described procedure maximal uptake of the dipeptide occur proximally, while free (23) and data expressed for animals of all ages on the basis of glycine has its highest influx levels in the distal ileum. weight. It is of interest that the same conclusions can be drawn also With the experimental procedure described above, both the when the values are expressed on the basis of tissue area. luminal and the serosal sides of the intestine were exposed to the Effects of a-L-Amino Acids and Peptides on Gly-Pro Influx. To incubation medium. However, preliminary experiments were done investigate the specificity of the Gly-Pro influx, we have measured under conditions in which only the serosal side was exposed: both it under conditions in which either a 40-fold excess of free amino glycyl-proline and glycine uptakes were always less than 5% of acids or a 10-fold excess of dipeptides was present.