Pfifigers Archiv European JoumN of Physiology 9 by Springer-Verlag 1978

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1 Pfligers Arch. 376, (1978) Pfifigers Archiv European JoumN of Physiology 9 by Springer-Verlag 1978 Concentrative Amino Acid Uptake at the Serosal Side of Colon Mucosa* E. Scharrer with the technical assistance of B. Amann Institut ffir Physiologic, Physiologische Chemic und Ernfihrungsphysiologie, Fachbereich Tiermedizin, Universitfit Miinchen, VeterinfirstraBe 13, D-8000 Mtinchen 22, Federal Republic of Germany Abstract. Uptake of the nonmetabolizable model amino acid 2-aminoisobutyric acid (= AIB) through the basolateral membrane into epithelial cells was studied in sheep colon stripped of serosa and muscle layers. Only the antiluminal surface of the mucosa was exposed to the incubation medium. Thus AIB entry into epithelial cells could only occur through the basolateral membrane. AIB was taken up by a saturable process against a high concentration gradient. AIB uptake was inhibited by other neutral amino acids but not by sugars. In a low Na + medium AIB uptake was impaired, indicating that active transport of amino acids through the basolateral membrane of colon epithelial cells is Na +-dependent. In the rat a saturable concentrative uptake of AIB through the basolateral membrane of colon epithelial cells has also been demonstrated. Concentrative uptake of amino acids through the basolateral membranes is probably important for the supply of colon epithelium with amino acids. Key words: Colon - Amino acid uptake - Basolateral membrane - Active transport - Sheep - Rat. pigs [8, 13], mice [10] and rats [2, 6, 10] with amino acids results in an intracellular accumulation of amino acids. This applies to L-phenylalanine in dogs, mice and rats [10], to L-methionine in new-born pigs [8, 13] and to L- valine [6] and L-proline [2] in rats. In further studies colon mucosa slices from humans [10] and guinea pigs [10] did not concentrate L- phenylalanine. Rat colon slices also did not accumulate L-alanine [3]. It is possible that the capability of colon mucosa to accumulate amino acids against a concentration gradient in most species is more closely related to amino acid metabolism and requirements of the tissue than to a specific mechanism concerned with the absorption of amino acids from the intestinal lumen [6]. In order to cover their amino acid requirements, colon epithelial cells might therefore possess specific transport mechanisms for concentrative amino acid uptake through the basolateral membrane. The aim of the present study was to investigate this possibility. The experiments were carried out with isolated sheep colon mucosa mounted so that only the antiluminal surface was exposed to the incubation medium. Some experiments were also performed with isolated rat colon mucosa. Introduction It is well known that in many species amino acids are not absorbed against a concentration gradient by the colon [10 3-6, 9]. Only in the dog [10] and new-born pig [7] a net transport of amino acids from the mucosal to the serosal side of isolated colon has been demonstrated [7, 10]. Furthermore some investigators have shown that incubating colon slices from dogs [11], new-born * Supported by Deutsche Forschungsgemeinschaft Methods and Material Lambs about 2 months old were used for the experiments. The upper colon was removed under anesthesia (1 mg Rompun 1 and 2.5mg Vetalar 2 per kg body weight i.m.) and transferred into oxygenated (OzCO2 = 955) cold Krebs Henseleit bicarbonate buffer. Thereupon the serosa ancl muscle layers were removed with a spatula. Histological examination indicated that the serosa and muscle layers were completely removed (Fig. 1). Then pieces of colon mucosa were mounted on plastic stoppers so that only the serosal surface was exposed to the incubation medium. The mucosa was attached to the 1 Bayer, Leverkusen 2 Parke Davis & Co., Miinchen $1.00

$246 Pfligers Arch. 376 (1978) Rubber stopper ( t \,@J Plastic stopper Rubber ring Colon mucosa medium Fig. 2.$

2 246 Pfligers Arch. 376 (1978) Rubber stopper ( t \,@J Plastic stopper Rubber ring Colon mucosa medium Fig. 2. of colon mucosa mounted so on a plastic stopper that only the serosal surface was exposed to the incubation medium Fig. 1 Histology of sheep colon mucosa stripped of serosa and muscle layers ( x 45) medium was measured in the same way. Quenching was similar for all samples. Therefore no quench corrections had to be made. AIB uptake into the epithelial cells was related to 1 g tissue wet weight. In addition the ratio between the AIB concentration of the intracellular fluid and the incubation medium (= [AIB]J[AIB]o was calculated as described previously [12]. For comparison some experiments were also carried out with rat colon mucosa in a similar manner. Adult Sprague Dawley rats (weight: g) obtained from Mns Rattus GmbH, Brunnthal, were used for these experiments. 14C-labelled AIB and 3H-labelled inulin were obtained from Amersham Buchler GmbH, Braunschweig. plastic stoppers with a ring of rubber fitting into a circular groove of the stopper. Then the preparations were incubated in 100ml centrifuge tubes containing 20 ml Krebs Henseleit bicarbonate buffer (Fig. 2). The tubes were filled with the appropriate gaseous phase (OzCOz = 955) and then closed with rubber stoppers and agitated (I 80 oscillationsmin) at 37 ~ C in an incubator. During the incubation the plastic stoppers with the colon mucosa were attached to the rubber stoppers with a thread. In this way the plastic stoppers could easily be taken Out from the tube at the end of the incubation. 14Clabelled 2-aminoisobutyric acid (AIB) was used as substrate. In order to correct for radioactivity present in the extracellular fluid, the incubation medium in addition contained 3H-labelled inulin (concentration: 0.02 mmol1). The tissue was first incubated for 15 min in Krebs Henseleit bicarbonate buffer before exposing it to the labelled amino acid and inulin. After the incubation the mucosa surrounded by the rubber ring was excised and weighed immediately. Both tissue water and 14C- and 3H-activity of samples were determined. The total tissue water was assessed by measuring the change in weight after drying part of the samples at 105 ~ C. For ~4C- and all-measurements the samples were oxidized in a Packard Sample Oxidizer (Model 306). Following the combustion process, this apparatus separates 14C-activity (COz) from 3H-activity (HzO). Thus the 14C- and 3H-activity of the samples could be assayed separately in aliquid scintillation spectrometer. Monophase 40 (Packard) was used as scintillation fluid for measuring 3H-activity. Carbo-Sorb (Packard) was employed as CO 2- absorber, and ~4C-activity was assayed using PermafluorV (Packard) as scintillation fluid. 14C- and 3H-activity of the incubation Results In Fig. 3 the time course of AIB uptake through the basolateral membrane into colon epithelium is shown. It is evident that AIB uptake was linear at least up to 16min. Because we wanted a substantial uptake, we chose 15 min for measuring initial uptake rates. For comparison AIB uptake through the luminal membrane of the colon epithelium is also presented in Fig. 3. In the latter experiments colon mucosa was mounted so only the luminal surface was exposed to the incubation medium. Intracellular AIB uptake was much lower in this case. In Fig. 4 the concentration dependence of AIB uptake through the basolateral membrane is shown. Apparently AIB is taken up by a saturable transport process. The Lineweaver Burk plot of these data shown in Fig. 5 indicates that the transport process follows Michaelis Menten type kinetics. From the Lineweaver Burk plot a K,~ of 311 mmol1 and a Vma x of 6.2 gmolg 9 15 min has been calculated for sheep colon mucosa. The

3 E. Scharrer: Amino Acid Uptake by Colon Mucosa 247 1"~ (, X -;SEM ~ OS ~ 1 (31 { (6) I (6) i i r min Fig. 3. Time course ofintraceilular uptake of AIB from the serosal (9 or luminal (0) side of sheep colon mucosa. AIB concentration: 1 mmol1. Number of preparations from two sheep in parenthesis (12) 1 correlation between [-~ and, L I v shown in Fig. 5 is significant (P < 0.01). This applies also to the other two linear plots of the Michaelis Menten function versus v and [S] versus ~). -[g] AIB apparently is taken up by active transport against a concentration gradient through the basolateral membrane of colon epithelial cells, as distribution ratios between the AIB concentration of the intracellular fluid and the incubation medium (= [AIBHAIB]o ) much higher than one were obtained at low AIB concentrations of the incubation medium (Table 1). This concentrative uptake of AIB was reduced by the presence of 2,4-dinitrophenol in the incubation medium by 23% (P<0.01). It is therefore in part dependent on the integrity of cellular energetic metabolism. In addition AIB transport through the basolateral membrane of the colon epithelium appears to be Nadependent, since AIB transport was impaired, if the Na-concentration of the Krebs-Henseleit buffer was.s "..0. E g &o. < -6 zo, E 1.o- I AIB concentration. rnmo[[ Fig. 4. Concentration dependence ofintracellular uptake of AIB from the serosal side of sheep colon mucosa. Number of preparations from 4 sheep in parenthesis Table 1. Distribution ratio of AIB between the intracellular fluid of sheep colon mucosa and the incubation medium (= [AIB]I[AIB]o). Only the serosal surface of the mucosa was exposed to the medium. AIB cone. of the medium: 1 mmol1 time (rain) [AIB]J[AIB]o _ 0.10" (27) b (3) (3) " 2 + S.E.M. b Number of preparations; 10 sheep were used for the experiments (8) " ~ i o--o Sheep ~. m ~ m (8) Y = i@o~ 0@15.~ ", Kin-- 6,8 rnmott " Vmax = 6.7 I..Irno[g-15 min Fig. 5 Lineweaver Burk plot of the concentration dependence of intracellular uptake of AIB from the serosal side of sheep and rat colon mucosa. Number of preparations from 4 sheep and 8 rats in parenthesis -o.~,,8, ~ I ~(i2) Km= 3.1 mmott ()2) (12) Vrnax = 6.3~m~ig,lSmin Q lmz, [ [~]

24g PflfigersArch. 376 (1978) E:z R -?- SEN (8 l 1; 2; 30 rain Fig. 6. Time course of intracellular uptake of AIB from the serosal side of rat colon mucosa. AIB concentration: 0.5 mmol1.

4 24g PflfigersArch. 376 (1978) E:z R -?- SEN (8 l 1; 2; 30 rain Fig. 6. Time course of intracellular uptake of AIB from the serosal side of rat colon mucosa. AIB concentration: 0.5 mmol1. Number of preparations from 7 rats in parenthesis The data obtained with rat colon mucosa are presented in Figs. 5 and 6 and in Table 2. The results are qualitatively similar to the corresponding sheep data. 1 For the rat colon mucosa the correlation between -- [s] and ~ (Fig. 5) was also significant (P < 0.001). This v again applies also to the other linear plots of the Michaelis Menten function. The affinity of the transport mechanism to AIB appears to be lower in the rat, because Km for AIB uptake (Fig. 5) was higher in the rat than in the sheep. Table 2. [AIB]i[AIB]o for rat colon mucosa exposed with the serosai surface to the incubation medium. AIB conc. of the medium: 0.5 retool1 time (min) [AIB]J[AIB] o a (6) b (6) _ (14) (8) _ (12) a 2 S.E.M. b Number of preparations; 7 rats were used for the experiments reduced to 30 mm by replacing NaCl with cholinechloride, KC1 of LiC1. In the low Na medium AIB uptake was 44% (replacement of NaC1 by cholinechloride), 52 %(replacement of NaC1 by LiC1), and 24 % (replacement of NaC1 by KC1) of the control values. The inhibition was significant in every case (P < 0.001). In this context it should be mentioned that cholinechloride (20 mmol1) per se did not inhibit AIB uptake (AIB concentration: 1 mmol1). AIB transport through the basolateral membrane was also inhibited by other neutral amino acids but not by sugars. In these experiments the concentration of AIB was 1 mmol1. In the presence of glycine (10mmol1) or L-leucine (10 mmol1) in the incubation medium AIB uptake was 47 % and 78 % of the control values, respectively. The inhibiting effect of both amino acids was significant (P< and P< 0.02, respectively). The presence of glucose (10mmol1) or 2-deoxyglucose (10 mmoll) in the incubation medium did not influence AIB uptake. Hence at least part of the natural neutral amino acids probably compete for the same transport mechanism. Discussion It has been shown in several species that colon epithelial cells possess the ability to accumulate amino acids against a concentration gradient [2, 6, 8-10, 13]. Because in most of these studies colon slices or colon mucosa slices have been used, amino acid uptake into epithelial cells could have happened through the luminal orand the basolateral membrane in these investigations. Our results demonstrate that the nonmetabolizable model amino acid AIB enters sheep colon epithelial cells through the basolateral membrane by an energy-dependent, saturable process, which is sodium-dependent. Since AIB uptake through the basolateral membrane was inhibited by other neutral amino acids but not by sugars, AIB apparently shares the same transport site with other neutral amino acids. As in our experiments AIB uptake into colon epithelia cells occurred from the antiluminal side, the amino acid requirement of colon epithelium is probably covered at least in part by amino acid uptake from the :blood side. This might also apply to other species, in, which a net transfer of amino acids from the mucosal to the serosal side of isolated colon could not be demonstrated, though there was an accumulation of amino acids in colon cells [2, 3, 5, 6]. This assumption is substantiated by the fact that we also could demonstrate an uptake of AIB against a concentration gradient through the basolateral membranes of rat colon epithelium. In some species such as the dog and pig (new-born) the situation may be different. In, this case both an accumulation of amino acids in colon cells and a net transfer of amino acids from the mucosal to the serosal side of the colon occurred [7, 10]. Therefore in these species in contrast to other species such as the rat, mouse and sheep, amino acid accumulation in colon ceils may at least in part be related to amino acid absorption. Since in our experiments AIB uptake through the basolateral membrane into sheep colon epithelium was

E. Scharrer: Amino Acid Uptake by Colon Mucosa 249 reduced markedly, when the NaC1 of the incubation medium was replaced by choline-chloride, KCI and LiC1, respectively, the sodium gradient across

5 E. Scharrer: Amino Acid Uptake by Colon Mucosa 249 reduced markedly, when the NaC1 of the incubation medium was replaced by choline-chloride, KCI and LiC1, respectively, the sodium gradient across the basolateral membrane could be implicated in the transport mechanism for amino acids in this membrane. In this case, an amino acid molecule and a sodium ion would form a ternary complex with a protein "carrier" for entry into the epithelial cell, in analogy with amino acid uptake through the brush border membrane into epithelial cells of the small intestine [12]. Acknowledgements. The author wishes to thank Prof. Dr. H.-G. Liebich, Lehrstuhl ffir Histologie und Embryologie, for the histological examination. References 1. Baillien, M., Schoffeniels, E. : Le transport actif d'acides amin+s au niveau de i'~pith61ium intestinal isol+ de Ia torme grecque. Biochim Biophys. Acta 53, (1961) 2. Batt, E. R., Schachter, D.: Developmental pattern of some intestinal transport mechanisms in newborn rats and mice. Am. J. Physiol. 216, (1969) 3. Binder, H. J. : Amino acid absorption from the colon. Biochim. Biophys. Acta 219, (1970) 4. Christensen, H. N., Feldman, B. H., Hastings, A. B.: Concentrative and reversible character of intestinal amino acid transport. Am. J. Physiol. 205, (1963) 5. Cordero, N., Wilson, T. H. : Comparison of transport capacity of small and large intestine. Gastroenterology 41, (1961) 6. Evered, D. H., Nunn, P. B. : Uptake of amino acids by mucosa of rat colon in vitro. Eur. J. Biochem. 4, (1968) 7. James, P. S., Smith, M. W. : Methionine transport by pig colonic mucosa measured during early post-natffl development. J. Physiol. (Lond.) 262, (1976) 8. Jarvis, L. G., Morgan, G., Smith, M. W., Wooding, F. B. P. : Cell replacement and changing transport function in the neonatal pig colon. J. Physiol. (Lond.) 273, (1977) 9. Nathans; D., Tapley, D. F., Ross, J. E.: Intestinal transport of amino acids studied in vitro with L-[131J]monoiodotyrosine. Biochim. Biophys. Acta 41, (1960) 10. Robinson, J. W. L., Luisier, A. L., Mirkovitch, V. : Transport of amino-acids and sugars by the dog colon mucos. Pflfigers Arch. 345, (1973) 11. Scharrer, E., Blatt, J.: In vitro-untersuchungen zur Aminosfiurenaufnahme in die Leber- und Muskelzelle beim Lamm. Zbl. Vet. Med. A 23, (1976) 12. Schultz, S. G., Curran, P. F. : Coupled transport of sodium and organic solutes. Physiol. Rev. 50, (1970) 13. Smith, M. W., James, P. S.: Amino acid transport by the helicoidal colon of the new-born pig. Biochim. Biophys. Acta 419, (1976) Received May 3, 1978

simple test of an amino acid's participation in an active transport D-fucose, L-glucose, oc-glucoheptose, L-fucose, D-mannose and

simple test of an amino acid's participation in an active transport D-fucose, L-glucose, oc-glucoheptose, L-fucose, D-mannose and 166 J. Physiol. (1966), 186, pp. 166-174 Printed in Great Britain EFFECT OF AMINO ACIDS ON SUGAR ABSORPTION BY J. T. HINDMARSH,* D. KILBY AND G. WISEMAN From the Department of Physiology, The University,