626 J. Physiol. (I956) I33, 626-630 ACTIVE TRANSPORT OF AMINO ACIDS BY SACS OF EVERTED SMALL INTESTINE OF THE GOLDEN HAMSTER (MESOCRICETUS AURATUS) BY G. WISEMAN From the Department of Physiology, University of Sheffield (Received 31 May 1956) The small intestine of the golden hamster (Mesocricetus auratus) has been shown to be able to transport the L-forms of proline, histidine and methionine against a concentration gradient and that they compete for the mechanism concerned (Wiseman, 1955). It was also shown that the L-forms of lysine and ornithine are not transported actively and have no effect on the active transport of proline, glycine, histidine and methionine when present in equimolecular amounts. In experiments with rat small intestine it was found that active transport of the L-forms of alanine, phenylalanine, methionine, histidine and isoleucine occurs but not of glutamic and aspartic acid (Wiseman, 1953). In 1952 Pinsky & Geiger published results of experiments in the rat in which it was shown that the rate of L-histidine absorption in vivo is decreased by L-tryptophan, and in 1954 Harper, Benton, Winje & Elvehjem showed that an excess of leucine in the diet prevents the normal utilization of isoleucine by the young rat. As an interpretation of the accumulated data it was suggested (Wiseman, 1955) that the mechanism for active transport of amino acids by the intestine is limited to the mono-amino-mono-carboxylic acids and that they compete with each other for this mechanism. The experiments described below were carried out in order to obtain data in regard to the ability of the hamster small intestine to transport actively threonine, alanine, serine, valine, hydroxyproline, phenylalanine, isoleucine, leucine and tryptophan. It is shown here that, apart from tryptophan, all these amino acids are transported actively, and the results of these experiments and those of Wiseman (1955) show that the amino acid best transported is proline and the descending order for the others is threonine, alanine, glycine, serine, valine, histidine, hydroxyproline, phenylalanine, isoleucine, leucine and methionine.
ACTIVE TRANSPORT OF AMINO ACIDS 627 METHODS Preparation of tissue. The method used for preparing and filling sacs of everted small intestine of the hamster was that described by Wilson & Wiseman (1954). Six sacs, each of about 3 cm length, were obtained from each small intestine (jejunum and ileum). At least three animals were used for each investigation of an amino acid. Measurement of initial and final volumes. The initial volume of fluid (serosal) introduced into the carefully drained sac of everted intestine (about 3 cm length) was recorded from the 1 ml. tuberculin syringe which was used for the introduction of the fluid. This serosal fluid volume was initially between 0*5 and 1.0 ml. The final volume of the serosal fluid was estimated by draining the sac of its fluid contents and weighing the fluid obtained. The increase in the volume of the serosal fluid during the experimental period was considered to be the volume of fluid transferred from mucosal to serosal sides. The volume of fluid (mucosal) in which the sac was placed at the beginning of the experimental period was 20 ml. Occasionally the serosal volume of a sac decreased during the experimental period. Such sacs were discarded. Experimental procedure. The sac, filled with a known volume of amino acid solution, was placed in a 150 ml. Erlenmeyer flask containing 20 ml. of the same amino acid solution as was used for filling the sac. The air in the flask was then replaced with a gas mixture of 5% CO2 + 95% 02 and the flask tightly stoppered. The flask and its contents were then kept at 370 C and continuously shaken for 1 hr by the use of a Warburg bath (rate of shaking 80 oscillations/min, amplitude 5 cm). At the end of 1 hr the sac was removed from the flask, its surface drained, and its fluid contents recovered and weighed. Samples of initial amino acid solution and final serosal and mucosal fluids were analysed for amino acid concentrations. A short length of thread ligature left at one end of the sac facilitates the removal of the sac from the flask. Amino acid solutions. The amino acids (all of the L-form) were commercial samples of chemically pure grade and were used without further purification. They were dissolved in a bicarbonate saline (Krebs & Henseleit, 1932) containing 0 3 % glucose and the solution was gassed with 5% CO2 + 95 % 02. The initial concentration of all the amino acid solutions was 20 mm. Dry weight. After removal of the serosal fluid the sacs were laid on Whatman no. 50 filter-paper, and the ends beyond the ligatures cut off and the ligature thread discarded. Excess surface fluid was then removed and the tissue dried for 2 hr at 110 C and weighed: the dry weights were of the order of 25-35 mg. Chemical estimations. Tryptophan was estimated by the colorimetric method ofspies & Chambers (1948). Alanine, phenylalanine, leucine, isoleucine, valine, serine and threonine were estimated by decarboxylation with a 10% (w/v) solution of chloramine-t in a citrate buffer of ph 2-5 and in the presence of 0.15 ml. 40% formaldehyde. The gas produced was measured manometrically by the use of conventional Warburg flasks and manometers and the final gas volume was measured after the reaction had been allowed to proceed for 20 min at 370 C. The rate of shaking of the flasks was 80 oscillations/min and the amplitude 5 cm. The presence of this amount of formalin has been shown to inhibit almost completely the production of N2 during the reaction of chloramine-t with amino acids and from NH3 present in the experimental solutions (Kemble & Macpherson, 1954). Table 1 gives the mole C02/mole amino acid obtained with standard solutions of the various amino acids and these recovery factors were applied accordingly. Control experiments. In some experiments sacs of everted intestine were filled with and incubated in bicarbonate-saline and glucose without added amino acid in order to determine the amount of chloramine-t reacting material which might be liberated from the tissue. After incubation for 1 hr no chloramine-t reacting material was found in the mucosal fluid from these sacs, but a small amount was found in the serosal fluid. The results of experiments in which amino acids were used were corrected to allow for this. Rate of transference and concentration gradients. The rate of accumulation of amino acid in the serosal fluid during the experimental period is given in pl./mg dry wt. of sac/hr, and is referred
628 G. WISEMAN to as the rate of transference. The concentration gradient is the ratio of the amino acid concentration in the serosal fluid to that in the mucosal fluid. Standard deviations were obtained using the formula for small samples. TABLE 1. Mole C02/mole amino acid obtained by decarboxylation of amino acids with chloramine-t at ph 2-5 Mole C02/mole Mole C02/mole Amino acid amino acid Amino acid amino acid Alanine 0-98 Phenylalanine 0 93 Hydroxyproline 0 95 Serine 0 95 Isoleucine 1-04 Threonine 0-91 Leucine 0.95 Valine 1-01 (Each flask contained: 1 ml. sample; 1 ml. M-citrate buffer ph 2-5; 0-15 ml. 40% aqueous formaldehyde; 1 ml. 10% (w/v) aqueous chloramine-t. Reaction time 20 min; temp. 370 C.) TABLE 2. Rates of transference of amino acids and concentration gradients developed by sacs of everted intestine Rate of transference Concentration gradient Amino acid (pll/mg dry wt./hr) developed No. of sacs Proline* 14-0+3-2 2-08±0-18 9 Threonine 12-0±2-7 1-90±0-28 14 Alanine 11-5±3-5 1 82+0-32 12 Glycine* 10-1±2-4 1 65±0 19 9 Serine 8-8±2-2 1-61±0-19 15 Valine 8-2±2-7 1-42±0-19 14 Histidine* 5-3±2-8 1-42+0-22 12 Hydroxyproline 6-3 1-8 1-36±0-14 12 Phenylalanine 5-4+2-2 1-24±0-11 11 Isoleucine 40+±1-2 1-19+0-09 13 Leucine 4 0+1-8 1.18±0-10 12 Methionine* 3-3±1-9 1-18±0-11 11 Tryptophan 1.1+0-9 0-90±0-05 7 (Initial concentration of each amino acid 20 mm inside and outside sac. Mucosal vol. 20 ml. Serosal vol. 0-5-1.0 ml. Gradients are expressed as ratio of amino acid concentration on serosal side to that on mucosal side. Figures shown are mean and standard deviation. Experimental period 1 hr, 370 C.) * These results are taken from Wiseman (1955) and are included in this table for the sake of completeness. The experiments were performed under identical conditions. RESULTS The recovery factors shown in Table 1 are in close agreement with those found by Kemble & Macpherson (1954) and except for phenylalanine and threonine show yields of CO2 reasonably close to theoretical. Table 2 gives the rates of transference of the amino acids and the concentration gradients developed by the sacs. To improve the usefulness of the table the results obtained by Wiseman (1955) for proline, glycine, histidine and methionine have been included (the experiments having been performed under the same conditions). All the amino acids except tryptophan were transferred against a concentration gradient. In the case of tryptophan the final concentration ratio was less than 1 owing to the increase in volume of the serosal fluid which occurs during the
ACTIVE TRANSPORT OF AMINO ACIDS 629 course of the experiment, while movement of tryptophan in the direction of the concentration gradient resulted in a small rate of transference of the amino acid. DISCUSSION In the above experiments all the amino acids except tryptophan were transported actively and the concentration gradients developed were within the range found by Wiseman (1955) for proline, glycine, histidine and methionine. That tryptophan should prove to be an exception was unexpected, especially in view of the results of Pinsky & Geiger (1952) who showed that it decreases the rate of histidine absorption in live rats, presumably competing for the same transporting mechanism. The reason for the inability of the intestine to transport tryptophan actively is not apparent. It seems unlikely that its relatively large molecular volume prevents its being transported actively as its rate of movement with the concentration gradient (i.e. passive diffusion) is greater than that found by Wiseman (1955) for the smaller molecules lysine and ornithine. A consideration of the concentration gradients developed and the molecular volumes of the amino acids suggests that there is no simple relationship between the two, although the amino acids with a relatively small molecular volume (e.g. glycine, alanine and serine) are amongst those best concentrated. The nitrogen atom in the aromatic ring of tryptophan has no basic properties and tryptophan must be classed as a mono-amino-mono-carboxylic acid; on the other hand, one of the nitrogen atoms in the aromatic ring of histidine (which is well concentrated) does have basic properties. Proline and hydroxyproline, both transported actively, have their nitrogen atom as part of a ring, but as it acts very much as it would were it in an open-chain secondary amine these amino acids must also be grouped with the simple mono-amino-monocarboxylic acids. The suggestion (Wiseman, 1955) that the mechanism for active transport of amino acids by the intestine is limited to the mono-aminomono-carboxylic acids should, therefore, be considered only as a general rule. Study of three-dimensional models of the amino acids has not led to any obvious relationship between a special molecular configuration and the degree to which an amino acid is concentrated. The rotation about bonds which occurs freely in most amino acids in solution makes any attempt to correlate degree of active transport with spatial arrangement almost impossible, especially in the absence of detailed and accurate knowledge of preferred configurations. SUMMARY 1. Sacs of everted small intestine of the hamster have been used to determine the concentration gradient developed during transference of amino acids from mucosal to serosal sides of the sac. The rates of such transference against a concentration gradient were also estimated.
630 G. WISEMAN 2. The sacs were found to transport actively threonine, alanine, serine, valine, hydroxyproline, phenylalanine, isoleucine and leucine. 3. Active transport of tryptophan did not occur. 4. The suggestion that the mechanism for active transport of amino acids by the intestine is limited to the mono-amino-mono-carboxylic acids should be considered only as a general rule. Part of the expense of this work was defrayed by grants from the Medical Research Fund of the University of Sheffield, and from the Medical Research Council. The technical assistance of Mr P. Price is gratefully acknowledged. The author wishes to thank Messrs L. Light and Co. (Colnbrook, Bucks) for generous gifts of amino acids. REFERENCES HARPER, A. E., BENTON, D. A., WINJE, M. E. & ELVEHJEM, C. A. (1954). Leucine-isoleucine antagonism in the rat. Arch. Biochem. Biophy8. 51, 523-524. KEMBLE, A. R. & MACPHERSON, H. T. (1954). Determination of monoamino monocarboxylic acids by quantitative paper chromatography. Biochem. J. 56, 548-555. KREBS, H. A. & HENSELEIT, K. (1932). Untersuchungen fiber die Hamstoffbildung im Tierkorper. Hoppe-Seyl. Z. 210, 33-66. PINSKY, J. & GEIGER, E. (1952). Intestinal absorption of histidine as influenced by tryptophan in the rat. Proc. Soc. exp. Biol., N. Y., 81, 55-57. SPIES, J. R. & CHAMBERS, D. C. (1948). Chemical determination of tryptophan. Analyt. Chem. 20, 30-39. WILSoN, T. H. & WISEMAN, G. (1954). The use of sacs of everted small intestine for the study of the transference of substances from mucosal to serosal surfaces. J. Phy8iol. 123, 116-125. WISEMAN, G. (1953). Absorption of amino-acids using an in vitro technique. J. Phy8iol. 120, 63-72. WISEMAN, G. (1955). Preferential transference of amino-acids from amino-acid mixtures by sacs of everted small intestine of the golden hamster (Mesocricetus auratue). J. Phy8iol. 127, 414-422.