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Multiple pathways for amino acid transport in brush border membrane vesicles isolated from the human fetal small intestine
GASTROENTEROLOGY
1991;100:1644-1652
Multiple Pathways for Amino Acid Transport in Brush Border Membrane Vesicles Isolated From the Human Fetal Small Intestine CHRISTIANE MALO Membrane University
Transport Research Group, Department of Montreal, Montreal, Quebec, Canada
The present study was undertaken to identify the different amino acid transport pathways present in the human small intestine during the early gestational period. The uptake time courses of neutral (L-leucine, L-alanine, L-methionine), acidic (L-glutamic and n-aspartic acids), basic (L-lysine), and imino (L-proline) acids have been studied in brush border membrane vesicles isolated from both proximal and distal parts of the human fetal small intestine. Both Na+-dependent and Na+-independent uptake pathways have been identified all along the small intestine. The Na’-dependent systems are as follows: (a) the NBB system for neutral amino acids such as L-leucine and L-alanine; (b) the PHE system for L-methionine; (c) the Xi system for L-glutamic and n-aspartic acids; and (d) the IMINO system for L-proline. The Na’-independent pathways are represented by the L system for most of the neutral amino acids and maybe L-proline and by the basic amino system y+ for L-lysine uptake. These results demonstrate that the different uptake pathways for transport of amino acids are present in the human fetal intestine and that their characteristics in terms of Na’ requirement and proximodistal activity gradient are already established in the early stages of the human development.
It
is actually well recognized that the transport of amino acids across the plasma membrane is a complex phenomenon that involves multiple carrier proteins. These different systems with overlapping specificities have been classified by Christensen (1,Z) in a wide variety of nonepithelial cells from different tissues and from various species. Amino acid transport has also been studied in rabbit (3,4), rat (5,6), mouse (7,8), bovine (9), pig (lO,ll), and guinea pig (12-14) small intestine as well as in rat kidney cortex
of Physiology,
Faculty
of Medicine,
(15-17). Our knowledge concerning ontogeny of these transport functions in the small intestine of different animal models has also been well summarized by Buddington and Diamond (18). From these studies, some species differences and organ specificities have been brought forward (1,18), thus rendering hazardous the extrapolation of these results to human tissues for which very sparse data have been obtained. In 1968, Levin et al. demonstrated that the tranfer of L-alanine in everted gut sacs from l4- to 21-week-old human fetuses is an electrogenie process (19). In 1986, Moriyama studied the transport of different amino acids in the human fetal small intestine and reported that both lysine and phenylalanine transport appears later than transport of alanine, leucine, taurine, and valine (20). These studies represent the sum of our knowledge on amino acid transport during the course of human development. On the other hand, few reports have been concerned with neutral, imino, and acidic amino acid transport in brush border membrane vesicles isolated from human adult small intestine (21-24). In 1977, Liicke et al. (21) showed that the transport of both L-alanine and L-phenylalanine is an Na’-dependent process. Similarly, both L-proline and glycine uptakes are stimulated by an Na’ gradient (22). In two other reports, kinetic studies of L-leucine and acidic amino acid transport were performed and, Michaelis constant (K,,,) values were estimated to be in the range of 0.39-0.5 mmol/L and 78-91 bmol/L for L-leucine (23) and L-glutamic acid (24) transport, respectively. Despite the fact that these reports are the only ones performed directly on human tissues, a large amount of information has been obtained from the study of
o 1991 by the American Gastroenterological 0016-5065/91/$3.00
Association
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1991
INTESTINAL AMINO ACIDS TRANSPORT
inborn defects of amino acid transport. At least 20 disorders of amino acid transport are known (25), and many of them have provided some potentially valuable clues about the mechanism involved. For example, in Hartnup disease, which involves intestinal and renal defects in transport of many neutral amino acids, the transport of imino acids such as glycine, proline, and hydroxyproline is not impaired, thus indicating the existence of at least one distinct transport system for these imino acids (2 5). The different amino acid transport systems have been classified according to various categories of amino acids such as neutral, basic, acidic, and imino acids, and both Na’-dependent and Na’-independent pathways have been described (2,26). In the brush border membrane of the enterocytes, at least six different transport systems have been identified (27): for the neutral amino acids, two Na’-dependent (NBB and Phe) and one Na’-independent (L) systems are present. The basic amino acids are taken up by the Na’-independent y+ system, the acidic amino acids by the Na’-dependent X, system, and the imino acids by an Na’-dependent imino system. Moreover, considering that no specific inhibitor is presently available and taking into account that “only occasionally does a natural amino acid serve as a defining substrate for a given transport system,” as stated by Christensen (l), it clearly appears that the study of amino acid transport characteristics represents a major challenge. However, given the importance of these functions from both a physiological and a pathological point of view, the study of the normal development of these amino acid transport systems in human small intestine should be helpful in defining the exact nature of the involved system. Recently, our laboratory has been concerned with the study of Na+-D-glucose cotransport systems in intestinal brush border membrane vesicles isolated from 17- to 26week-old human fetuses (28-30). We have shown that a proximodistal gradient of transport activity is already present in the early stages of gestation (28), and we have further characterized these systems by showing that two distinct pathways are present in the fetal jejunum although a single system was involved in the ileum (2930). More recently, we have shown that there is only a single system for Na+-D-glucose cotransport in the adult human jejunum (31), which points out that some important modifications of these functions occur in the course of the human development. Therefore, extrapolations of the data obtained from adult studies to fetal or neonatal organs could lead to erroneous conclusions. Considering that no complete study of the different pathways for amino acid transport in the course of human intestinal development is presently available, we have oriented our efforts to the identifi-
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cation of these systems to provide some basis to their subsequent characterization. In this study, we have studied the uptake time course of four different classes of amino acids (neutral, acidic, basic, and imino) along the human fetal small intestine, and we have defined the properties of these different transport pathways in terms of their Na’ requirement. Our data show that different amino acid transport systems are functional in the human fetal small intestine and that a proximodistal gradient of transport capacity is already established at an early stage of the human development.
Materials and Methods Chemicals All salts and chemicals for buffer preparation were of the highest purity available. L-[“Qglutamic acid, L-[“‘Cl alanine,L-[3H]methionine,L-[3H]proline,L-[3H]lysine,D-[3H]aspartic acid, and D-[I-‘%]mannitOl were purchased from Amersham Canada Ltd. (Oakville, Ontario, Canada); L-[‘Clleucine and D-[l-3H(n)]mannitol were purchased from New England Nuclear Corporation (Mississaugua, Ontario, Canada).
Preparation of Brush Border Membrane Vesicles Fresh, 17- to 20-week-old normal human fetal small intestines were kindly provided by Dr. Jean Michaud from Ste-Justine Hospital where social abortions have been performed. This research was approved by the ethical committee of the Faculty of Medicine, University of Montreal, in accordance with the guidelines defined by the Medical Research Council of Canada. The jejunum and the ileum were separated, and the mucosa of each segment was scraped, weighed, and frozen in liquid nitrogen until the day of experiment (28). An average of eight tissues were pooled for each transport experiment. The brush border membranes were purified by CaCl, precipitation (32), and vesicles were prepared as previously described (28). Based on sucrase (apical membrane marker) and sodium-potassium-stimulated ATPase (Na’,K’ -ATPase, basolateral membrane marker) activities, enrichment factors of 17-22-fold over the homogenate were routinely obtained and contamination by basolateral membranes was always < 50%(28).
Transport Studies Time-course studies of amino acid uptake were performed using the rapid filtration technique of Hopfer et al. (33). Freshly purified brush border membrane vesicles were resuspended to a final protein concentration of 6-8 mg/mL in the resuspension buffer containing 50 mmol/L Tris-HEPES buffer (pH 7.5), 0.1 mmol/L MgSO,, 250 mmol/L KCl, and 100 mmol/L mannitol. An aliquot of the brush border membrane vesicle preparation (100 FL) was added to the incubation medium (400 uL) kept at room temperature to start the transport experiments. The time courses of
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CHRISTIANE MALO
amino acid uptakes were performed under the following three different experimental conditions: (a) under an Na’gradient condition (200 mmol/L out]; (b) in the absence of Na’; and (c) in the presence of saturating concentration of unlabeled substrate (50 mmol/L). The final concentrations in the incubation media were as follows: 50 mmol/L TrisHEPES buffer, pH 7.5; 0.1 mmol/L MgSO,; 200 mmol/L NaCl; 50 mmol/L KC1 or 250 mmol/L KCl; 100 mmol/L mannitol or 50 mmol/L mannitol and 50 mmol/L unlabeled substrate; and 50 p,mol/L radiolabeled substrate. At time intervals, aliquots were taken from the incubation mixture and poured into 1 mL quenched ice-cold stop solution for which the composition was adjusted to match the final concentrations of the different species in the incubation medium. The quenched mixture was then filtered on a prewetted and chilled 0.45 pmol/L nitrocellulose filter (SM 11306; Sartorius GmbH, Gottingen, Germany) and washed with 4 mL of nonradioactive ice-cold stop solution. Filters were dissolved in 5 mL Filter Counter [United Technologies Packard, Mississauga, Ontario, Canada), and radioactivity was counted using a Minaxi Tri-Carb Series 4000, model 4450 scintillation counter (United Technologies Packard). All data were corrected for the dead space as measured with radiolabeled mannitol added to the ice-cold stop solution. Results are expressed as nanomoles solute uptake per milligram protein. Results Time Courses of Neutral Amino Acid Uptake
thionine
The uptake has been
of L-leucine, L-alanine, measured as a function
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both proximal and distal parts of the human fetal small intestine. As shown in Figure 1, there was a transient overshoot of L-leucine uptake in the presence of an Na+-gradient (200 mmol/L out), with a peak 1 minute in both proximal (Figure lA) and around distal (Figure 1B) parts of the small intestine, although the level of uptake was lower and the overshoot was less pronounced in the ileum. The uptake recorded in the absence of Na’ in the incubation medium was higher than the level of the passive diffusion of leucine, obtained by measuring the uptake of radiolabeled substrate in the presence of 50 mmol/L unlabeled leucine. A similar behavior was observed in the ileum, thus indicating the presence of both Na’-dependent and Na’-independent pathways for L-leucine in the human fetal small intestine. The time course of L-alanine in both jejunal and ileal brush border membrane vesicles (Figure 2A and B) was similar to the one observed for L-leucine, but the overshoot phenomena was clearly less marked in this case and the maximum intravesicular accumulation was lower. Comparing the relative proportion of uptake by the different pathways (Table l),one can appreciate that the Na+-dependent pathway contributes for 64% to 79% of the total uptake of L-leucine and L-alanine in both intestinal regions. Moreover, L-methionine gave a completely different profile. As shown in Figure 3, there was some evidence for both Na’-dependent and Na’-independent uptake pathways, but the nonspecific uptake of L-me-
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Figure I. Uptake time course of L-leucine in brush border membrane vesicles isolated from both proximal (A)and distal (8) parts of the human fetal small intestine. The vesicles were resuspended in 50 mmol/L. Tris-HEPES buffer, pH 7.5: 0.1 mmol/L MgSO,; 250 mmol!L KCl; and 100 mmol/L mannitol. The final concentrations in the incubation media were 50 mmol/L Tris-HEPES buffer, pH 7.5; 0.1 mmol/L MgSO,; 200 mmol/L NaCl; 50 mmol/L KC1 (0) or 250 mmohL KC1 (0); 100 mmol/L mannitol (0, 0) or 50 mmohL mannitol; and 50 mmol/L unlabeled substrate (A) and 50 ~moliL QC]leucine. Points shown are individual data from one preparation of vesicles obtained from a pool of eight different tissues.
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B
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thionine represented the major component (see Table 1). This behavior was noticed in both jejunal and ileal brush border membrane vesicles, and the proximodistal gradient was clearly perceptible. Time Courses of Acidic Amino Acid Uptake
Both L-glutamic (Figure 4) and D-aspartic (Figure 5) acid uptakes have been evaluated under different experimental conditions. Contrary to what has been observed for the neutral amino acids, the uptake of both acidic amino acids occurred mainly by the Na+-dependent pathway (Table 1). There was no evidence for Na’-independent uptake, and passive diffusion was very low in both jejunal and ileal vesicles. It should also be noted that the level of L-glutamic intravesicular accumulation was very high
Table I. Percentage of Amino Acid Uptake by Different Path ways Uptake pathways Na’ dependent
L-Leu L-Ala L-Meth L-Glu ~-Asp
Nonspecific
P
D
P
D
P
D
79 64 19 95 90
67 66 17 92 86
11 9 9 3 4 20
22 14 15 5 6 22
10 27 72 2 6 80
11 20 68 3 8 76
23
28
14
12
63
60
L-Lys L-Pro
Na+ independent
The relative proportion (%) of the different uptake components has been calculated from the uptake values recorded at 0.5 minutes under different experimental conditions. Each amino acid has been tested at the same concentration (50 kmol/L) as described in the text. P. proximal small intestine; D, distal small intestine.
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and that the proximodistal gradient was very prominent for both acidic amino acids. Time Course of Basic Amino Acid Uptake
L-Lysine uptake has been evaluated all along the human fetal small intestine. The data reported in Figure 6 indicate that L-lysine was taken up by both the Na+-independent pathway and the nonspecific uptake component in the two intestinal regions. Here again, the proximodistal gradient was clearly noticeable. There was no stimulation of L-lysine uptake in the presence of an Na’ gradient (200 mmol/L out). Time Course of Imino Acid Uptake
The uptake of L-proline was very low in the two intestinal regions (Figure 7A and B), but a stimulation of L -proline uptake in the presence of an Na’ gradient can be observed. In the complete absence of Na’ in the incubation medium, there was also a small but significant accumulation of L-proline over the nonspecific uptake level; these data indicate the coexistence of both Nat-dependent and Na’-independent pathways for L-proline uptake during the early gestational period. Discussion
The absorption of amino acids from the lumen of the small intestine to the blood involves different mechanisms such as passive diffusion through the paracellular and cellular routes and mediated transport through apical and basolateral membranes. The use of brush border membrane vesicles for transport
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me-
is broadly selective for most neutral amino acids. Accordingly, the uptake of the neutral amino acids L-leucine and L-alanine has been found to be both Nat-dependent and Na’-independent in jejunal and ileal brush border membrane vesicles isolated from the human fetuses, thus suggesting the presence of these active pathways in the developing human small intestine. A similar pattern for neutral amino acid uptake has already been demonstrated in mouse small intestine (7). A typical overshoot phenomenon can be observed for L-leucine uptake in the presence of an Na’ gradient in both regions of the small intestine whereas this overshoot is less pronounced for L-alanine in the jejunum and almost completely absent in the ileum. This behavior might be because the entry of L-alanine is slower than the Na’ movement so that the
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Figure 4. Uptake time course of L-[“C]glutamic acid uptake in jejunum (A) and ileum (B) of the human fetal small intestine. Both the resuspension and incubation media were as described in the legend of Figure 1.
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Figure 5. Uptake time course of oj3H]aspartic acid in proximal (A)and distal (B) small intestine. See Figure 1 for details.
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driving force required for the accumulation of L-alanine cannot be fully exerted. This slow movement of L-alanine is in accordance with the observation that the peak of alanine accumulation is observed around 3 minutes whereas L-leucine overshoot peaks around 1 minute. However, the relative proportion both of L-leucine and L-alanine taken up by NBB and L transport pathways is similar when evaluated at the same concentration (Table l), which suggests that these amino acids share the same transport pathways. L-Methionine is another neutral amino acid for which a different Na’-dependent transport pathway, the PHE system, has been identified. This pathway is responsible for the uptake of phenylalanine and methionine in the intestinal brush border membrane (26,27). Contrary to what has been observed for
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leucine and alanine, the presence of an Na’ gradient does not induce a large stimulation of methionine uptake although the level observed was higher than the one obtained in the presence of K’ (Figure 3). The time course of L-methionine uptake under different experimental conditions suggests the presence of both Na’-dependent PHE and Na’-independent L systems in the human fetal small intestine. Such Na’dependent and Na’-independent uptake pathways have been described in everted sacs of mouse jejunum (7) and in bovine jejunal and ileal brush border membrane vesicles (9). Furthermore, no overshoot phenomenon can be observed for the Na’-dependent uptake, as previously observed with phenylalanine in human adult brush border membrane vesicles (21). The relative proportion of uptake by the Na’-
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Figure 6. Time course of t$H]lysine uptake in proxima1 (A)and distal (B)small intestine. Experimental conditions and symbols were as described in the legend of Figure 1.
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independent L pathway is similar to the one recorded for leucine and alanine. The characteristics of acidic amino acid transport systems have been studied in rabbit (3), rat (5), and human adult (24) intestinal brush border membrane vesicles, and all these studies have shown that Na’ is indispensably required for this uptake. The apical membrane transport system responsible for the uptake of both L-glutamic and n-aspartic acids has been classified as X, (2,26). This system appears very active in the human fetal small intestine, especially for L-glutamic acid, and the uptake time course observed is similar to the one shown previously for rabbit (3) and human adult (24) tissues. It is interesting to note that the experimental conditions chosen for these assays were highly favorable for the uptake because a K’ gradient [in > out) was present along with the inwardly directed Na’ gradient. The stimulation of Na’-L-glutamic acid cotransport by internal K’ has already been demonstrated in rabbit (3) and human (24) intestinal brush border membrane vesicles and could probably account for the hightransport activity observed in the human fetus. There is no uptake over the passive diffusion level in the absence of Na+ in the incubation medium, indicating that these amino acids do not enter the vesicles by the Na’-independent amino acid pathway. This behavior differs completely from the one observed for the basic amino acid L-lysine for which Na’ does not induce further stimulation of the uptake. The mediated transport of L-lysine seems to be entirely assumed by the Na’-independent y+ system, specific for the basic amino acids such as lysine, arginine, and cationic amino acids (2,26,27). The same pattern of uptake is observed in the ileum and the proximodistal gradient of activity is clearly established. These re-
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Figure 7. Uptake time course of L-[3H]proline in jejunum (A) and~&um (B) of human fetuses. See Figure 1 for details.
sults differ from those obtained from mouse small intestine (7) and bovine brush border membrane vesicles (9); in these studies, both Na’ -dependent and Na’-independent uptake pathways have been found. In everted sacs of mouse jejunum (7), the Na’dependent uptake of lysine was completely inhibited by leucine, thus suggesting that lysine can be taken up by the Na’-dependent neutral amino acid system in mouse small intestine. On the other hand, the Na’-dependent uptake of L-lysine in bovine brush border membrane vesicles (9) was almost undetectable at lysine concentrations <0.3 mmol/L, a substrate concentration much higher than the one used in our experiment (50 tJ,mol/L). Furthermore, total lysine uptake was higher in the ileum compared with the jejunum, a pattern that was not observed in the human fetal small intestine [Figure 6). Therefore, these discrepancies can be partly explained by both species differences and variable experimental conditions. In contrast to these results, it has been clearly shown that the L-lysine uptake occurs through a single saturable Na’-independent system plus a significant diffusional component in rat renal brush border membrane vesicles (17). Similarly, the transport of cationic amino acids in renal cell lines LLC-PK, (pig kidney) and MDCK (dog kidney) has been shown to occur through the Na’-independent y+ system, although different behavior for the Na’-dependent inhibition of lysine uptake by homoserine and glutamine has been reported in these two cell lines (34). Further characterization in terms of substrate specificity is needed to define the nature of the system responsible for lysine transport in human small intestine. Finally, the last system studied concerns the IMINO pathway which is recognized as Na’ dependent and highly specific for proline and imino acids (2,26,27).
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INTESTINAL AMINO ACIDS TRANSPORT
In the small intestine of human fetus, this system does not appear very active although a significant stimulation of proline uptake is observed in the presence of an inwardly directed Na’ gradient as previously demonstrated in human adult intestinal brush border membrane vesicles (22). The uptake of L-proline in the absence of Na’ in the incubation medium is slightly greater than that obtained in the presence of 50 mmol/L unlabeled substrate, thus indicating that L-proline can be partially taken up by an Na’-independent uptake pathway which can be tentatively identified as the brush border membrane L system. As summarized in Table 1, the seven different amino acids studied could represent each of the six different uptake pathways identified in the intestinal brush border membrane. The main characteristics of these systems in terms of Na’ requirement are already set in the human fetus. The Na’-dependent systems present include the following: (a) the NBB system for the neutral amino acids such as L-leucine and L-alanine which is very active and represents the major component of the total uptake for these two amino acids; (b) the PHE system which is responsible for the uptake of the neutral amino acids phenylalanine and methionine is also present all along the human fetal small intestine although less active than the NBB system; (c) the Xi8 system, specific for acidic amino acids such as L-glutamic and o-aspartic acids is one of the most efficient system in terms of concentrative capacity of the amino acids into the vesicles; and (d) the IMINO system which is well represented by L-proline uptake, is also present at the brush border membrane level at this early stage of human intestinal development. The Na’-independent systems identified are the L system, which is broadly specific and assumes the Na’-independent carrier-mediated uptake of neutral amino acids and maybe of L-proline, and the basic amino system y+, which is responsible for the total carrier-mediated uptake of proline. In all cases, a proximodistal gradient of activity has been noted. A nonspecific uptake component has also been measured in all cases, although large variations have been observed from one amino acid to the other. This component is minimal for acidic amino acids, a result in accordance with previous reports (3,5,24) and also consistent with the fact that a negatively charged molecule does not tend to diffuse easily into the membrane lipid phase. For L-leucine and L-alanine, this nonspecific uptake component is slightly higher but still at low level. On the other hand, this uptake component appears very large for L-methionine, L-lysine, and L-proline, an observation that deserves some comments. This nonspecific uptake component, which cannot be inhibited by 50 mmol/L unlabeled substrate, could represent either passive diffusion of
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these amino acids into the vesicles or nonspecific binding of the radiolabeled substrate to the vesicles. As pointed out by Christensen (35), a major contribution to total uptake of amino acids by simple diffusion appears quite unlikely considering the low solubility of free amino acids in the membrane lipid phase. On the other hand, a nonspecific binding of amino acids to the brush border membrane has to be considered because all the uptake time curves may actually not go through zero and may intercept the y-axis at a higher value; in the absence of multiple time points at very short time intervals, no precise determination of the initial rate of uptake nor of the binding component can be performed adequately. However, such binding can be expected, at least for a basic amino acid such as lysine, but is more difficult to explain in the case of the neutral methionine although the sulfur linked in a thioether bond can account for this behavior. For proline, a binding component to the external membrane has already been reported (13,16).Nevertheless, the presence of a nonspecific uptake component does not modify our conclusion as to the presence of both Na’-dependent and Na’-independent pathways for the uptake of L-methionine and L-proline all along the human fetal intestine as well as to the existence of only an Na+-independent pathway for L-lysine transport, but an underestimation of the relative proportion of the total uptake through the carrier-mediated pathways could be expected. In conclusion, the results obtained on amino acid uptake over the length of the human fetal intestine indicate the early appearance of all the systems already identified in intestinal brush border membrane vesicles. These data also show that the characteristics of these systems in terms of Na’ dependency are well established within the first months of gestation. The present report establishes some basis to the study of the ontogeny of human amino acid transport functions. However, a comparative evaluation of these systems in terms of kinetic characteristics and substrate specificities is needed in both fetal and adult tissues before a developmental pattern of these systems can be obtained. Such studies would certainly be helpful for a better understanding of the clinical symptoms associated with perturbations of these important human functions.
References 1. Christensen HN. On the development of amino acid transport systems. Fed Proc 1973;32:19-28. 2. Christensen HN. On the strategy of kinetic discrimination of amino acid transport systems. J’Membr Biol 1985;84:97-103. 3. Berteloot A. Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Biochim Biophys Acta 1984;775:129-140. 4. Stevens B, Wright EM. Substrate specificity of the intestinal
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5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18. 19.
20.
21.
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brush-border prolineisodium (IMINO) transporter. J Membr Biol 1985;87:27-34. Corcelli A, Prezioso G, Palmieri F, Storelli C. Electroneutral Na+/dicarboxylic amino acid cotransport in rat intestinal brush border membrane vesicles. Biochim Biophys Acta 1982;689:97105. Younoszai MK, Smith C, Finch MH. Comparison of in vitro jejunal uptake of L-valine and L-lysine in the rat during maturation. J Pediatr Gastroenterol Nutr 1985;4:992-997. Karasov W, Solberg D, Carter S, Hughes M, Phan D, Zollman F, Diamond. Uptake pathways for amino acids in mouse intestine. Am J Physiol 1986;251:G501-G508, Batt ER, Schachter D. Developmental pattern of some intestinal transport mechanisms in newborn rats and mice. Am J Physiol 1969;216:1064-1068. Wilson JW, Webb KE. Lysine and methionine transport by bovine jejunal and ileal brush border membrane vesicles. J Anim Sci 1990;68:504-514. Seplilveda FV, Smith MW. Different mechanisms for neutral amino acid uptake by new-born pig colon. J Physiol 1979;286: 479-490. Wolffram S, Eggenberger E, Sharrer E. Kinetics of n-glucose and L-leucine transport into sheep and pig intestinal brush border membrane vesicles. Comp Biochem Physiol [A] 1986;84:589593. Munck BG. Imino acid transport across the brush-border membrane of the guinea-pig small intestine. Biochim Biophys Acta 1984;770:35-39. Hayashi K, Kawasaki T. The characteristic changes of amino acid transport during development in brush border membrane vesicles of the guinea pig ileum. Biochim Biophys Acta 1982; 691:83-90. Himukai H, Konno T, Hoshi T. Age-dependent change in intestinal absorption of dipeptides and their constituent amino acids in the guinea-pig. Pediatr Res 1980;14:1272-1275. Baerlocher KE, Striver CR, Mohyuddin F. The ontogeny of amino acid transport in rat kidney. I. Effect on distribution ratios and intracellular metabolism of proline and glycine. Biochim Biophys Acta 1971;249:353-363. Medow MS, Roth KS, Goldmann DR, Ginkinger K, Hsu BY, Segal S. Developmental aspects of proline transport in rat renal brush border membranes. Proc Nat1 Acad Sci USA 1986;83: 7561-7564. McNamara PD, Rea CT, Segal S. Lysine uptake by rat renal brush-border membranevesicles. Am J Physiol1986;251:F734F742. Buddington RK, Diamond JM. Ontogenic development of intestinal nutrient transporters. Ann Rev Physiol 1989;51:601-619. Levin RJ, Koldovsky 0, Hoskova J, Jirsova V, Uher J. Electrical activity across human foetal intestine associated with absorption processes. Gut 1968;9:206-213. Moriyama IS. Development of fetal organs and adaptation to extrauterine life. Acta Obstet Gynecol Jpn Engl Ed 1986;38: 1227-1237. Liicke H, Haase W, Murer H. Amino acid transport in brushborder-membrane vesicles isolated from human small intestine. Biochem J 1977;168:529-532.
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22. Rajendran VM, Ansari SA, Harig JM, Adams MB, Khan AH, Ramaswamy K. Transport of glycyl-L-proline by human intestinal brush border membrane vesicles. Gastroenterology 1985;89: 1298-1304. 23. Harig JM, Barry JA, Rajendran VM, Soergel KH, Ramaswamy K. n-glucose and L-leucine transport by human intestinal brushborder membrane vesicles. Am J Physiol1989;256:G618-G623. 24. Rajendran VM, Harig JM, Adams MB, Ramaswamy K. Transport of acidic amino acids by human jejunal brush-border membrane vesicles. Am J Physiol 1987;252:G33-G39, 25. Wellner D, Meister A. A survey of inborn errors of amino acid metabolism and transport in man. Ann Rev Biochem 1981;50: 911-968. 26. Berteloot A, Malo C. Maladies membranaires de l’intestin et du rein. Med Sci 1985;1:427-434. 27. Stevens BR, Kaunitz JD, Wright EM. Intestinal transport of amino acids and sugars: advances using membrane vesicles. Ann Rev Physiol 1984;46:417-433, 28. Malo C, Berteloot A. Proximo-distal gradient of Na+-dependent n-glucose transport activity in the brush border membrane vesicles from the human fetal small intestine. FEBS Lett 1987;220:201-205. 29. Malo C. Kinetic evidence for heterogeneity in Na’-u-glucose transport systems in the normal human fetal small intestine. Biochim Biophys Acta 1988;938:181-188. 30. Malo C. Separation of two distinct Na’-n-glucose cotransport systems in the human fetal jejunum by means of their differential specificity for 3-0-methylglucose. Biochim Biophys Acta 1990;1022:8-16. 31. Malo C, Berteloot A. Analysis of kinetic data in transport studies: new insights from kinetic studies of Na’-o-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus. J Membr Biol 1991 (in press). 32. Schmitz J, Preiser H, Maestracci D, Ghosh BK, Cerda JJ, Crane RK. Purification of the human intestinal brush border membrane. Biochim Biophys Acta 1973;323:98-112. 33. Hopfer U, Nelson K, Perrotto J, Isselbacher KJ. Glucose transport in isolated brush-border membrane from rat small intestine. J Biol Chem 1973;248:25-32. 34. Sepulveda FV, Pearson JD. Cationic amino acid transport by two renal epithelial cell lines: LLC-PK, and MDCK cells. J Cell Physiol 1985;123:144-150, 35. Christensen HN. Distinguishing amino acid transport systems of a given cell or tissue. Methods Enzymol 1989;173:576-616.
Received August 14,199O. Accepted November 20,199O. Address requests for reprints to: Christiane Malo, Ph.D., Membrane Transport Research Group, Department of Physiology, Faculty of Medicine, University of Montreal, Montreal, Quebec, H3C 3J7 Canada. This work was supported by a grant from the Medical Research Council of Canada (MA-8923). The author is supported by a scolarship from the “Fends de la Recherche en Sante du Quebec.” The author greatly acknowledges Dr. Jean Michaud for providing human tissues and L. Lessard for technical assistance.
1991;100:1644-1652
Multiple Pathways for Amino Acid Transport in Brush Border Membrane Vesicles Isolated From the Human Fetal Small Intestine CHRISTIANE MALO Membrane University
Transport Research Group, Department of Montreal, Montreal, Quebec, Canada
The present study was undertaken to identify the different amino acid transport pathways present in the human small intestine during the early gestational period. The uptake time courses of neutral (L-leucine, L-alanine, L-methionine), acidic (L-glutamic and n-aspartic acids), basic (L-lysine), and imino (L-proline) acids have been studied in brush border membrane vesicles isolated from both proximal and distal parts of the human fetal small intestine. Both Na+-dependent and Na+-independent uptake pathways have been identified all along the small intestine. The Na’-dependent systems are as follows: (a) the NBB system for neutral amino acids such as L-leucine and L-alanine; (b) the PHE system for L-methionine; (c) the Xi system for L-glutamic and n-aspartic acids; and (d) the IMINO system for L-proline. The Na’-independent pathways are represented by the L system for most of the neutral amino acids and maybe L-proline and by the basic amino system y+ for L-lysine uptake. These results demonstrate that the different uptake pathways for transport of amino acids are present in the human fetal intestine and that their characteristics in terms of Na’ requirement and proximodistal activity gradient are already established in the early stages of the human development.
It
is actually well recognized that the transport of amino acids across the plasma membrane is a complex phenomenon that involves multiple carrier proteins. These different systems with overlapping specificities have been classified by Christensen (1,Z) in a wide variety of nonepithelial cells from different tissues and from various species. Amino acid transport has also been studied in rabbit (3,4), rat (5,6), mouse (7,8), bovine (9), pig (lO,ll), and guinea pig (12-14) small intestine as well as in rat kidney cortex
of Physiology,
Faculty
of Medicine,
(15-17). Our knowledge concerning ontogeny of these transport functions in the small intestine of different animal models has also been well summarized by Buddington and Diamond (18). From these studies, some species differences and organ specificities have been brought forward (1,18), thus rendering hazardous the extrapolation of these results to human tissues for which very sparse data have been obtained. In 1968, Levin et al. demonstrated that the tranfer of L-alanine in everted gut sacs from l4- to 21-week-old human fetuses is an electrogenie process (19). In 1986, Moriyama studied the transport of different amino acids in the human fetal small intestine and reported that both lysine and phenylalanine transport appears later than transport of alanine, leucine, taurine, and valine (20). These studies represent the sum of our knowledge on amino acid transport during the course of human development. On the other hand, few reports have been concerned with neutral, imino, and acidic amino acid transport in brush border membrane vesicles isolated from human adult small intestine (21-24). In 1977, Liicke et al. (21) showed that the transport of both L-alanine and L-phenylalanine is an Na’-dependent process. Similarly, both L-proline and glycine uptakes are stimulated by an Na’ gradient (22). In two other reports, kinetic studies of L-leucine and acidic amino acid transport were performed and, Michaelis constant (K,,,) values were estimated to be in the range of 0.39-0.5 mmol/L and 78-91 bmol/L for L-leucine (23) and L-glutamic acid (24) transport, respectively. Despite the fact that these reports are the only ones performed directly on human tissues, a large amount of information has been obtained from the study of
o 1991 by the American Gastroenterological 0016-5065/91/$3.00
Association
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1991
INTESTINAL AMINO ACIDS TRANSPORT
inborn defects of amino acid transport. At least 20 disorders of amino acid transport are known (25), and many of them have provided some potentially valuable clues about the mechanism involved. For example, in Hartnup disease, which involves intestinal and renal defects in transport of many neutral amino acids, the transport of imino acids such as glycine, proline, and hydroxyproline is not impaired, thus indicating the existence of at least one distinct transport system for these imino acids (2 5). The different amino acid transport systems have been classified according to various categories of amino acids such as neutral, basic, acidic, and imino acids, and both Na’-dependent and Na’-independent pathways have been described (2,26). In the brush border membrane of the enterocytes, at least six different transport systems have been identified (27): for the neutral amino acids, two Na’-dependent (NBB and Phe) and one Na’-independent (L) systems are present. The basic amino acids are taken up by the Na’-independent y+ system, the acidic amino acids by the Na’-dependent X, system, and the imino acids by an Na’-dependent imino system. Moreover, considering that no specific inhibitor is presently available and taking into account that “only occasionally does a natural amino acid serve as a defining substrate for a given transport system,” as stated by Christensen (l), it clearly appears that the study of amino acid transport characteristics represents a major challenge. However, given the importance of these functions from both a physiological and a pathological point of view, the study of the normal development of these amino acid transport systems in human small intestine should be helpful in defining the exact nature of the involved system. Recently, our laboratory has been concerned with the study of Na+-D-glucose cotransport systems in intestinal brush border membrane vesicles isolated from 17- to 26week-old human fetuses (28-30). We have shown that a proximodistal gradient of transport activity is already present in the early stages of gestation (28), and we have further characterized these systems by showing that two distinct pathways are present in the fetal jejunum although a single system was involved in the ileum (2930). More recently, we have shown that there is only a single system for Na+-D-glucose cotransport in the adult human jejunum (31), which points out that some important modifications of these functions occur in the course of the human development. Therefore, extrapolations of the data obtained from adult studies to fetal or neonatal organs could lead to erroneous conclusions. Considering that no complete study of the different pathways for amino acid transport in the course of human intestinal development is presently available, we have oriented our efforts to the identifi-
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cation of these systems to provide some basis to their subsequent characterization. In this study, we have studied the uptake time course of four different classes of amino acids (neutral, acidic, basic, and imino) along the human fetal small intestine, and we have defined the properties of these different transport pathways in terms of their Na’ requirement. Our data show that different amino acid transport systems are functional in the human fetal small intestine and that a proximodistal gradient of transport capacity is already established at an early stage of the human development.
Materials and Methods Chemicals All salts and chemicals for buffer preparation were of the highest purity available. L-[“Qglutamic acid, L-[“‘Cl alanine,L-[3H]methionine,L-[3H]proline,L-[3H]lysine,D-[3H]aspartic acid, and D-[I-‘%]mannitOl were purchased from Amersham Canada Ltd. (Oakville, Ontario, Canada); L-[‘Clleucine and D-[l-3H(n)]mannitol were purchased from New England Nuclear Corporation (Mississaugua, Ontario, Canada).
Preparation of Brush Border Membrane Vesicles Fresh, 17- to 20-week-old normal human fetal small intestines were kindly provided by Dr. Jean Michaud from Ste-Justine Hospital where social abortions have been performed. This research was approved by the ethical committee of the Faculty of Medicine, University of Montreal, in accordance with the guidelines defined by the Medical Research Council of Canada. The jejunum and the ileum were separated, and the mucosa of each segment was scraped, weighed, and frozen in liquid nitrogen until the day of experiment (28). An average of eight tissues were pooled for each transport experiment. The brush border membranes were purified by CaCl, precipitation (32), and vesicles were prepared as previously described (28). Based on sucrase (apical membrane marker) and sodium-potassium-stimulated ATPase (Na’,K’ -ATPase, basolateral membrane marker) activities, enrichment factors of 17-22-fold over the homogenate were routinely obtained and contamination by basolateral membranes was always < 50%(28).
Transport Studies Time-course studies of amino acid uptake were performed using the rapid filtration technique of Hopfer et al. (33). Freshly purified brush border membrane vesicles were resuspended to a final protein concentration of 6-8 mg/mL in the resuspension buffer containing 50 mmol/L Tris-HEPES buffer (pH 7.5), 0.1 mmol/L MgSO,, 250 mmol/L KCl, and 100 mmol/L mannitol. An aliquot of the brush border membrane vesicle preparation (100 FL) was added to the incubation medium (400 uL) kept at room temperature to start the transport experiments. The time courses of
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CHRISTIANE MALO
amino acid uptakes were performed under the following three different experimental conditions: (a) under an Na’gradient condition (200 mmol/L out]; (b) in the absence of Na’; and (c) in the presence of saturating concentration of unlabeled substrate (50 mmol/L). The final concentrations in the incubation media were as follows: 50 mmol/L TrisHEPES buffer, pH 7.5; 0.1 mmol/L MgSO,; 200 mmol/L NaCl; 50 mmol/L KC1 or 250 mmol/L KCl; 100 mmol/L mannitol or 50 mmol/L mannitol and 50 mmol/L unlabeled substrate; and 50 p,mol/L radiolabeled substrate. At time intervals, aliquots were taken from the incubation mixture and poured into 1 mL quenched ice-cold stop solution for which the composition was adjusted to match the final concentrations of the different species in the incubation medium. The quenched mixture was then filtered on a prewetted and chilled 0.45 pmol/L nitrocellulose filter (SM 11306; Sartorius GmbH, Gottingen, Germany) and washed with 4 mL of nonradioactive ice-cold stop solution. Filters were dissolved in 5 mL Filter Counter [United Technologies Packard, Mississauga, Ontario, Canada), and radioactivity was counted using a Minaxi Tri-Carb Series 4000, model 4450 scintillation counter (United Technologies Packard). All data were corrected for the dead space as measured with radiolabeled mannitol added to the ice-cold stop solution. Results are expressed as nanomoles solute uptake per milligram protein. Results Time Courses of Neutral Amino Acid Uptake
thionine
The uptake has been
of L-leucine, L-alanine, measured as a function
and L-meof time in
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both proximal and distal parts of the human fetal small intestine. As shown in Figure 1, there was a transient overshoot of L-leucine uptake in the presence of an Na+-gradient (200 mmol/L out), with a peak 1 minute in both proximal (Figure lA) and around distal (Figure 1B) parts of the small intestine, although the level of uptake was lower and the overshoot was less pronounced in the ileum. The uptake recorded in the absence of Na’ in the incubation medium was higher than the level of the passive diffusion of leucine, obtained by measuring the uptake of radiolabeled substrate in the presence of 50 mmol/L unlabeled leucine. A similar behavior was observed in the ileum, thus indicating the presence of both Na’-dependent and Na’-independent pathways for L-leucine in the human fetal small intestine. The time course of L-alanine in both jejunal and ileal brush border membrane vesicles (Figure 2A and B) was similar to the one observed for L-leucine, but the overshoot phenomena was clearly less marked in this case and the maximum intravesicular accumulation was lower. Comparing the relative proportion of uptake by the different pathways (Table l),one can appreciate that the Na+-dependent pathway contributes for 64% to 79% of the total uptake of L-leucine and L-alanine in both intestinal regions. Moreover, L-methionine gave a completely different profile. As shown in Figure 3, there was some evidence for both Na’-dependent and Na’-independent uptake pathways, but the nonspecific uptake of L-me-
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Figure I. Uptake time course of L-leucine in brush border membrane vesicles isolated from both proximal (A)and distal (8) parts of the human fetal small intestine. The vesicles were resuspended in 50 mmol/L. Tris-HEPES buffer, pH 7.5: 0.1 mmol/L MgSO,; 250 mmol!L KCl; and 100 mmol/L mannitol. The final concentrations in the incubation media were 50 mmol/L Tris-HEPES buffer, pH 7.5; 0.1 mmol/L MgSO,; 200 mmol/L NaCl; 50 mmol/L KC1 (0) or 250 mmohL KC1 (0); 100 mmol/L mannitol (0, 0) or 50 mmohL mannitol; and 50 mmol/L unlabeled substrate (A) and 50 ~moliL QC]leucine. Points shown are individual data from one preparation of vesicles obtained from a pool of eight different tissues.
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B
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thionine represented the major component (see Table 1). This behavior was noticed in both jejunal and ileal brush border membrane vesicles, and the proximodistal gradient was clearly perceptible. Time Courses of Acidic Amino Acid Uptake
Both L-glutamic (Figure 4) and D-aspartic (Figure 5) acid uptakes have been evaluated under different experimental conditions. Contrary to what has been observed for the neutral amino acids, the uptake of both acidic amino acids occurred mainly by the Na+-dependent pathway (Table 1). There was no evidence for Na’-independent uptake, and passive diffusion was very low in both jejunal and ileal vesicles. It should also be noted that the level of L-glutamic intravesicular accumulation was very high
Table I. Percentage of Amino Acid Uptake by Different Path ways Uptake pathways Na’ dependent
L-Leu L-Ala L-Meth L-Glu ~-Asp
Nonspecific
P
D
P
D
P
D
79 64 19 95 90
67 66 17 92 86
11 9 9 3 4 20
22 14 15 5 6 22
10 27 72 2 6 80
11 20 68 3 8 76
23
28
14
12
63
60
L-Lys L-Pro
Na+ independent
The relative proportion (%) of the different uptake components has been calculated from the uptake values recorded at 0.5 minutes under different experimental conditions. Each amino acid has been tested at the same concentration (50 kmol/L) as described in the text. P. proximal small intestine; D, distal small intestine.
NOTE.
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and that the proximodistal gradient was very prominent for both acidic amino acids. Time Course of Basic Amino Acid Uptake
L-Lysine uptake has been evaluated all along the human fetal small intestine. The data reported in Figure 6 indicate that L-lysine was taken up by both the Na+-independent pathway and the nonspecific uptake component in the two intestinal regions. Here again, the proximodistal gradient was clearly noticeable. There was no stimulation of L-lysine uptake in the presence of an Na’ gradient (200 mmol/L out). Time Course of Imino Acid Uptake
The uptake of L-proline was very low in the two intestinal regions (Figure 7A and B), but a stimulation of L -proline uptake in the presence of an Na’ gradient can be observed. In the complete absence of Na’ in the incubation medium, there was also a small but significant accumulation of L-proline over the nonspecific uptake level; these data indicate the coexistence of both Nat-dependent and Na’-independent pathways for L-proline uptake during the early gestational period. Discussion
The absorption of amino acids from the lumen of the small intestine to the blood involves different mechanisms such as passive diffusion through the paracellular and cellular routes and mediated transport through apical and basolateral membranes. The use of brush border membrane vesicles for transport
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CHRISTIANE MALO
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nine in the incubation dium.
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TIME (mid studies circumvents many of the problems encountered with intact epithelial tissues (27) and allows the study of carrier-mediated transport of both facilitated and active types at the apical membrane level. We have already demonstrated the validity of this approach for Na+-D-glucose cotransport systems in both jejunum and ileum of human fetuses (28-30). In the present report, we have used the same preparation to investigate the uptake capacity of the human fetal small intestine for four different classes of amino acids representing the six different transport pathways already identified in the small intestine (26,27). The apical Na’-dependent NBB system is responsible for the uptake of most neutral amino acids in the small intestine, whereas the Na’-independent L system, also present at the brush border membrane level,
me-
is broadly selective for most neutral amino acids. Accordingly, the uptake of the neutral amino acids L-leucine and L-alanine has been found to be both Nat-dependent and Na’-independent in jejunal and ileal brush border membrane vesicles isolated from the human fetuses, thus suggesting the presence of these active pathways in the developing human small intestine. A similar pattern for neutral amino acid uptake has already been demonstrated in mouse small intestine (7). A typical overshoot phenomenon can be observed for L-leucine uptake in the presence of an Na’ gradient in both regions of the small intestine whereas this overshoot is less pronounced for L-alanine in the jejunum and almost completely absent in the ileum. This behavior might be because the entry of L-alanine is slower than the Na’ movement so that the
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Figure 4. Uptake time course of L-[“C]glutamic acid uptake in jejunum (A) and ileum (B) of the human fetal small intestine. Both the resuspension and incubation media were as described in the legend of Figure 1.
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INTESTINAL AMINO ACIDS TRANSPORT
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Figure 5. Uptake time course of oj3H]aspartic acid in proximal (A)and distal (B) small intestine. See Figure 1 for details.
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driving force required for the accumulation of L-alanine cannot be fully exerted. This slow movement of L-alanine is in accordance with the observation that the peak of alanine accumulation is observed around 3 minutes whereas L-leucine overshoot peaks around 1 minute. However, the relative proportion both of L-leucine and L-alanine taken up by NBB and L transport pathways is similar when evaluated at the same concentration (Table l), which suggests that these amino acids share the same transport pathways. L-Methionine is another neutral amino acid for which a different Na’-dependent transport pathway, the PHE system, has been identified. This pathway is responsible for the uptake of phenylalanine and methionine in the intestinal brush border membrane (26,27). Contrary to what has been observed for
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leucine and alanine, the presence of an Na’ gradient does not induce a large stimulation of methionine uptake although the level observed was higher than the one obtained in the presence of K’ (Figure 3). The time course of L-methionine uptake under different experimental conditions suggests the presence of both Na’-dependent PHE and Na’-independent L systems in the human fetal small intestine. Such Na’dependent and Na’-independent uptake pathways have been described in everted sacs of mouse jejunum (7) and in bovine jejunal and ileal brush border membrane vesicles (9). Furthermore, no overshoot phenomenon can be observed for the Na’-dependent uptake, as previously observed with phenylalanine in human adult brush border membrane vesicles (21). The relative proportion of uptake by the Na’-
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Figure 6. Time course of t$H]lysine uptake in proxima1 (A)and distal (B)small intestine. Experimental conditions and symbols were as described in the legend of Figure 1.
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GASTROENTEROLOGY
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independent L pathway is similar to the one recorded for leucine and alanine. The characteristics of acidic amino acid transport systems have been studied in rabbit (3), rat (5), and human adult (24) intestinal brush border membrane vesicles, and all these studies have shown that Na’ is indispensably required for this uptake. The apical membrane transport system responsible for the uptake of both L-glutamic and n-aspartic acids has been classified as X, (2,26). This system appears very active in the human fetal small intestine, especially for L-glutamic acid, and the uptake time course observed is similar to the one shown previously for rabbit (3) and human adult (24) tissues. It is interesting to note that the experimental conditions chosen for these assays were highly favorable for the uptake because a K’ gradient [in > out) was present along with the inwardly directed Na’ gradient. The stimulation of Na’-L-glutamic acid cotransport by internal K’ has already been demonstrated in rabbit (3) and human (24) intestinal brush border membrane vesicles and could probably account for the hightransport activity observed in the human fetus. There is no uptake over the passive diffusion level in the absence of Na+ in the incubation medium, indicating that these amino acids do not enter the vesicles by the Na’-independent amino acid pathway. This behavior differs completely from the one observed for the basic amino acid L-lysine for which Na’ does not induce further stimulation of the uptake. The mediated transport of L-lysine seems to be entirely assumed by the Na’-independent y+ system, specific for the basic amino acids such as lysine, arginine, and cationic amino acids (2,26,27). The same pattern of uptake is observed in the ileum and the proximodistal gradient of activity is clearly established. These re-
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Figure 7. Uptake time course of L-[3H]proline in jejunum (A) and~&um (B) of human fetuses. See Figure 1 for details.
sults differ from those obtained from mouse small intestine (7) and bovine brush border membrane vesicles (9); in these studies, both Na’ -dependent and Na’-independent uptake pathways have been found. In everted sacs of mouse jejunum (7), the Na’dependent uptake of lysine was completely inhibited by leucine, thus suggesting that lysine can be taken up by the Na’-dependent neutral amino acid system in mouse small intestine. On the other hand, the Na’-dependent uptake of L-lysine in bovine brush border membrane vesicles (9) was almost undetectable at lysine concentrations <0.3 mmol/L, a substrate concentration much higher than the one used in our experiment (50 tJ,mol/L). Furthermore, total lysine uptake was higher in the ileum compared with the jejunum, a pattern that was not observed in the human fetal small intestine [Figure 6). Therefore, these discrepancies can be partly explained by both species differences and variable experimental conditions. In contrast to these results, it has been clearly shown that the L-lysine uptake occurs through a single saturable Na’-independent system plus a significant diffusional component in rat renal brush border membrane vesicles (17). Similarly, the transport of cationic amino acids in renal cell lines LLC-PK, (pig kidney) and MDCK (dog kidney) has been shown to occur through the Na’-independent y+ system, although different behavior for the Na’-dependent inhibition of lysine uptake by homoserine and glutamine has been reported in these two cell lines (34). Further characterization in terms of substrate specificity is needed to define the nature of the system responsible for lysine transport in human small intestine. Finally, the last system studied concerns the IMINO pathway which is recognized as Na’ dependent and highly specific for proline and imino acids (2,26,27).
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INTESTINAL AMINO ACIDS TRANSPORT
In the small intestine of human fetus, this system does not appear very active although a significant stimulation of proline uptake is observed in the presence of an inwardly directed Na’ gradient as previously demonstrated in human adult intestinal brush border membrane vesicles (22). The uptake of L-proline in the absence of Na’ in the incubation medium is slightly greater than that obtained in the presence of 50 mmol/L unlabeled substrate, thus indicating that L-proline can be partially taken up by an Na’-independent uptake pathway which can be tentatively identified as the brush border membrane L system. As summarized in Table 1, the seven different amino acids studied could represent each of the six different uptake pathways identified in the intestinal brush border membrane. The main characteristics of these systems in terms of Na’ requirement are already set in the human fetus. The Na’-dependent systems present include the following: (a) the NBB system for the neutral amino acids such as L-leucine and L-alanine which is very active and represents the major component of the total uptake for these two amino acids; (b) the PHE system which is responsible for the uptake of the neutral amino acids phenylalanine and methionine is also present all along the human fetal small intestine although less active than the NBB system; (c) the Xi8 system, specific for acidic amino acids such as L-glutamic and o-aspartic acids is one of the most efficient system in terms of concentrative capacity of the amino acids into the vesicles; and (d) the IMINO system which is well represented by L-proline uptake, is also present at the brush border membrane level at this early stage of human intestinal development. The Na’-independent systems identified are the L system, which is broadly specific and assumes the Na’-independent carrier-mediated uptake of neutral amino acids and maybe of L-proline, and the basic amino system y+, which is responsible for the total carrier-mediated uptake of proline. In all cases, a proximodistal gradient of activity has been noted. A nonspecific uptake component has also been measured in all cases, although large variations have been observed from one amino acid to the other. This component is minimal for acidic amino acids, a result in accordance with previous reports (3,5,24) and also consistent with the fact that a negatively charged molecule does not tend to diffuse easily into the membrane lipid phase. For L-leucine and L-alanine, this nonspecific uptake component is slightly higher but still at low level. On the other hand, this uptake component appears very large for L-methionine, L-lysine, and L-proline, an observation that deserves some comments. This nonspecific uptake component, which cannot be inhibited by 50 mmol/L unlabeled substrate, could represent either passive diffusion of
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these amino acids into the vesicles or nonspecific binding of the radiolabeled substrate to the vesicles. As pointed out by Christensen (35), a major contribution to total uptake of amino acids by simple diffusion appears quite unlikely considering the low solubility of free amino acids in the membrane lipid phase. On the other hand, a nonspecific binding of amino acids to the brush border membrane has to be considered because all the uptake time curves may actually not go through zero and may intercept the y-axis at a higher value; in the absence of multiple time points at very short time intervals, no precise determination of the initial rate of uptake nor of the binding component can be performed adequately. However, such binding can be expected, at least for a basic amino acid such as lysine, but is more difficult to explain in the case of the neutral methionine although the sulfur linked in a thioether bond can account for this behavior. For proline, a binding component to the external membrane has already been reported (13,16).Nevertheless, the presence of a nonspecific uptake component does not modify our conclusion as to the presence of both Na’-dependent and Na’-independent pathways for the uptake of L-methionine and L-proline all along the human fetal intestine as well as to the existence of only an Na+-independent pathway for L-lysine transport, but an underestimation of the relative proportion of the total uptake through the carrier-mediated pathways could be expected. In conclusion, the results obtained on amino acid uptake over the length of the human fetal intestine indicate the early appearance of all the systems already identified in intestinal brush border membrane vesicles. These data also show that the characteristics of these systems in terms of Na’ dependency are well established within the first months of gestation. The present report establishes some basis to the study of the ontogeny of human amino acid transport functions. However, a comparative evaluation of these systems in terms of kinetic characteristics and substrate specificities is needed in both fetal and adult tissues before a developmental pattern of these systems can be obtained. Such studies would certainly be helpful for a better understanding of the clinical symptoms associated with perturbations of these important human functions.
References 1. Christensen HN. On the development of amino acid transport systems. Fed Proc 1973;32:19-28. 2. Christensen HN. On the strategy of kinetic discrimination of amino acid transport systems. J’Membr Biol 1985;84:97-103. 3. Berteloot A. Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Biochim Biophys Acta 1984;775:129-140. 4. Stevens B, Wright EM. Substrate specificity of the intestinal
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brush-border prolineisodium (IMINO) transporter. J Membr Biol 1985;87:27-34. Corcelli A, Prezioso G, Palmieri F, Storelli C. Electroneutral Na+/dicarboxylic amino acid cotransport in rat intestinal brush border membrane vesicles. Biochim Biophys Acta 1982;689:97105. Younoszai MK, Smith C, Finch MH. Comparison of in vitro jejunal uptake of L-valine and L-lysine in the rat during maturation. J Pediatr Gastroenterol Nutr 1985;4:992-997. Karasov W, Solberg D, Carter S, Hughes M, Phan D, Zollman F, Diamond. Uptake pathways for amino acids in mouse intestine. Am J Physiol 1986;251:G501-G508, Batt ER, Schachter D. Developmental pattern of some intestinal transport mechanisms in newborn rats and mice. Am J Physiol 1969;216:1064-1068. Wilson JW, Webb KE. Lysine and methionine transport by bovine jejunal and ileal brush border membrane vesicles. J Anim Sci 1990;68:504-514. Seplilveda FV, Smith MW. Different mechanisms for neutral amino acid uptake by new-born pig colon. J Physiol 1979;286: 479-490. Wolffram S, Eggenberger E, Sharrer E. Kinetics of n-glucose and L-leucine transport into sheep and pig intestinal brush border membrane vesicles. Comp Biochem Physiol [A] 1986;84:589593. Munck BG. Imino acid transport across the brush-border membrane of the guinea-pig small intestine. Biochim Biophys Acta 1984;770:35-39. Hayashi K, Kawasaki T. The characteristic changes of amino acid transport during development in brush border membrane vesicles of the guinea pig ileum. Biochim Biophys Acta 1982; 691:83-90. Himukai H, Konno T, Hoshi T. Age-dependent change in intestinal absorption of dipeptides and their constituent amino acids in the guinea-pig. Pediatr Res 1980;14:1272-1275. Baerlocher KE, Striver CR, Mohyuddin F. The ontogeny of amino acid transport in rat kidney. I. Effect on distribution ratios and intracellular metabolism of proline and glycine. Biochim Biophys Acta 1971;249:353-363. Medow MS, Roth KS, Goldmann DR, Ginkinger K, Hsu BY, Segal S. Developmental aspects of proline transport in rat renal brush border membranes. Proc Nat1 Acad Sci USA 1986;83: 7561-7564. McNamara PD, Rea CT, Segal S. Lysine uptake by rat renal brush-border membranevesicles. Am J Physiol1986;251:F734F742. Buddington RK, Diamond JM. Ontogenic development of intestinal nutrient transporters. Ann Rev Physiol 1989;51:601-619. Levin RJ, Koldovsky 0, Hoskova J, Jirsova V, Uher J. Electrical activity across human foetal intestine associated with absorption processes. Gut 1968;9:206-213. Moriyama IS. Development of fetal organs and adaptation to extrauterine life. Acta Obstet Gynecol Jpn Engl Ed 1986;38: 1227-1237. Liicke H, Haase W, Murer H. Amino acid transport in brushborder-membrane vesicles isolated from human small intestine. Biochem J 1977;168:529-532.
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22. Rajendran VM, Ansari SA, Harig JM, Adams MB, Khan AH, Ramaswamy K. Transport of glycyl-L-proline by human intestinal brush border membrane vesicles. Gastroenterology 1985;89: 1298-1304. 23. Harig JM, Barry JA, Rajendran VM, Soergel KH, Ramaswamy K. n-glucose and L-leucine transport by human intestinal brushborder membrane vesicles. Am J Physiol1989;256:G618-G623. 24. Rajendran VM, Harig JM, Adams MB, Ramaswamy K. Transport of acidic amino acids by human jejunal brush-border membrane vesicles. Am J Physiol 1987;252:G33-G39, 25. Wellner D, Meister A. A survey of inborn errors of amino acid metabolism and transport in man. Ann Rev Biochem 1981;50: 911-968. 26. Berteloot A, Malo C. Maladies membranaires de l’intestin et du rein. Med Sci 1985;1:427-434. 27. Stevens BR, Kaunitz JD, Wright EM. Intestinal transport of amino acids and sugars: advances using membrane vesicles. Ann Rev Physiol 1984;46:417-433, 28. Malo C, Berteloot A. Proximo-distal gradient of Na+-dependent n-glucose transport activity in the brush border membrane vesicles from the human fetal small intestine. FEBS Lett 1987;220:201-205. 29. Malo C. Kinetic evidence for heterogeneity in Na’-u-glucose transport systems in the normal human fetal small intestine. Biochim Biophys Acta 1988;938:181-188. 30. Malo C. Separation of two distinct Na’-n-glucose cotransport systems in the human fetal jejunum by means of their differential specificity for 3-0-methylglucose. Biochim Biophys Acta 1990;1022:8-16. 31. Malo C, Berteloot A. Analysis of kinetic data in transport studies: new insights from kinetic studies of Na’-o-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus. J Membr Biol 1991 (in press). 32. Schmitz J, Preiser H, Maestracci D, Ghosh BK, Cerda JJ, Crane RK. Purification of the human intestinal brush border membrane. Biochim Biophys Acta 1973;323:98-112. 33. Hopfer U, Nelson K, Perrotto J, Isselbacher KJ. Glucose transport in isolated brush-border membrane from rat small intestine. J Biol Chem 1973;248:25-32. 34. Sepulveda FV, Pearson JD. Cationic amino acid transport by two renal epithelial cell lines: LLC-PK, and MDCK cells. J Cell Physiol 1985;123:144-150, 35. Christensen HN. Distinguishing amino acid transport systems of a given cell or tissue. Methods Enzymol 1989;173:576-616.
Received August 14,199O. Accepted November 20,199O. Address requests for reprints to: Christiane Malo, Ph.D., Membrane Transport Research Group, Department of Physiology, Faculty of Medicine, University of Montreal, Montreal, Quebec, H3C 3J7 Canada. This work was supported by a grant from the Medical Research Council of Canada (MA-8923). The author is supported by a scolarship from the “Fends de la Recherche en Sante du Quebec.” The author greatly acknowledges Dr. Jean Michaud for providing human tissues and L. Lessard for technical assistance.