Characteristics of an Amino Acid Transport System in Rat Liver for Glutamine, Asparagine, Histidine, and Closely Related Analogs*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Val No. 9, Issue of May 10, pp Printed in U.S.A. Characteristics of an Amino Acid Transport System in Rat Liver for Glutamine, Asparagine, Histidine,
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Val No. 9, Issue of May 10, pp Printed in U.S.A. Characteristics of an Amino Acid Transport System in Rat Liver for Glutamine, Asparagine, Histidine, and Closely Related Analogs* (Received for publication, October 9, 1979) Michael S. Kilberg, Mary E. Handlogten, and Halvor N. Christensen$ From the Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan amino acid from ASC, as discussed elsewhere (12, 13). Our older experiments did not establish, however, that Systems A and ASC account for all the uptake of glutamine and asparagine in red blood cells and the Ehrlich cell. Systems A, ASC, Na+-dependent system (, N ) and L participate vigorously in the uptake of neutral amino acids in the hepatocyte (12, 14, E), so a priori glutamine migration into that cell seemed well provided for. In the present paper we report that the rat hepatocyte In the rat hepatocyte, whether freshly separated or in primary culture, we do not find L-glutamine entry by Systems A and ASC as seen in cells previously studied. Instead the mediated entry of glutamine appears to occurexclusivelyby a apparently specific to amino acid amides and L-histidine; however, a portion of asparagine uptake occurs by System A. The simplest evidence for the separateness of the added system is the failure of model substrates for System A (e.g. N-methylalanine) to inhibit glutamine uptake significantly, and the failure of glutamine to inhibit the uptake of L-cysteine, model substrate for System ASC, at least in this cell. As is the case for cysteine, glutamine inhibits transport by System A (although not competitively), even though showing no transport by that system. Our finding confirms an earlier inference that glutamine uptake by this cell may follow a route not taken by alanine or serine, and explains the apparently erroneous companion inference that glutamine also shares a route with these two amino acids. Its uptake has now been characterized to show a series of differences from Systems A and ASC. Especially significant in view of the importance of glu- tamine metabolism are an insensitivity of the new system to stimulation by either insulin or glucagon, and its distinct enhancement (not as large as that for System A) on starvation of the cells with respect to amino acids. Hence, a second system has been found to show adaptive regulation. shows a third Na -dependent transport system which appears to be specific to glutamine, asparagine, histidine, and a few close unnatural homologs, and which accounts for most or all of the mediated uptake of glutamine by that cell. Its charac- terization here represents a confirmation of a remarkable foresight by Joseph et al. (16) that the interactions they saw among alanine, serine, and glutamine were consistent with the uptake of part of the glutamine by a route different from those that might serve principally for alanine and serine. Their experiments did not, however, establish such a transport system, nor indeed systematically limit uptake of their test amino acids to any single route. We will use the provisional abbreviation System N for the new transport activity described here, taking note that nitrogen occurs in the side chains of its natural substrates, and that the letter n occurs in the abbreviations, Gln and Asn. McGivan has agreed with us that System N is a useful designation. This abbreviation may also avoid exaggerated evocations of the sort that sometimes trouble the abbreviations A, L, and ASC (13). The circumstance that no transport of glutamine by Systems A and ASC could be shown for this cell, that MeAIB transport does not occur by System ASC or the new system, and that cysteine We have previously shown that included among the trans- uptake is restricted to System ASC, has facilitated their port systems mediating the passage of neutral a-amino acids differentiation. Glutamine can therefore serve as a model into and out of eukaryotic cells, investigated mainly with the substrate (although of course not as an unmetabolizable sub- Ehrlich ascites tumor cell and various red blood cells (1-ll), strate) for the new system, It does not serve as a model are a wide range, Na -independent system designated L, and inhibitor for System N, however, because like cysteine it is an at least two wide range, Na -dependent systems designated A effective inhibitor of System A, although in both cases the and ASC. In our studies in Ehrlich cells, rabbit reticulocytes, inhibition is not competitive. This action presumably explains and pigeon erythrocytes, asparagine and glutamine were found the inference of Joseph et al. (E), not confirmed by us, that to act as substrates for System ASC (9, 10) (also for Systems glutamine also enters by a route shared with alanine and A and L in the Ehrlich cell), which surprised us somewhat serine. Asparagine is not specific enough to System N to serve because other branched chain amino acids had previously as a model substrate. The discrimination of System N from failed to show unequivocal ASC substrate action. That sur- Systems A and ASC has been extended by demonstration of prise is decreased by the newer appreciation that the presence several other differentiating characteristics. System N proves of a branched aliphatic chain does not necessarily exclude an to be the fist Na+-dependent transport system, beyond Sys- * tem A (17), to show adaptive regulation, ie. stimulation when This work was supported by Grant HD01233 from the Institute of Child Health and Human Development, National Institutes of the hepatocyte is starved for amino acids, a finding of special Health, United States Public Health Service, and from a pilot project interest if one recalls the intense metabolic importance of under the Diabetes Research and Training Center (Grant glutamine and its quantitative pre-eminence among circulat- IPfNAM20572). The costs of publication of this article were defrayed ing amino acids. In contrast to System A, System N showed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. M. S. Kilberg, M. E. Handlogten, and H. N. Christensen, manu- $ To whom correspondence should be addressed. script in preparation 4012 Added Neutral Amino Acid Transport System in Rat Liver no stimulation by insulin or glucagon. These features add to Amino acid transport into hepatocytes after 24 h in culture was the clarity of differentiation of the three systems. measured by adding 2 ml of the uptake medium at 37 C to each dish for the desired interval of time followed by removal of the medium by EXPERIMENTAL PROCEDURES aspiration and five quick washes with ice-cold phosphate-buffered saline, ph 7.4. The cells were then removed by scraping the dish with Isolation and Culturing of Hepatocytes-Male Sprague-Dawley a rubber policeman in the presence of1.5 ml of0.2 N NaOH. rats (140 to 180 g) were anesthetized by intraperitoneal injection of Aliquots were taken for determination of both radioactivity and pentobarbital (7.5 mg/100 g of body weight). After exposure of the protein. Protein determinations were by the method of Lowry et al. viscera, the right renal vein was ligated and the inferior vena cava (20). Data are expressed in picomoles or nanomoles of test amino acid cannulated. Retrograde perfusion was begun at 7 ml/min with Swim's per mg of protein per unit time. The procedures for scintillation s-77 medium, ph 7.4, containing 25 mm NaHC03,50 p~ L-cystine, 5.8 counting were as described previously (21). pg/ml of streptomycin, 62.5 pg/ml of penicillin, and 0.1% BSA.' As Material.~--'~C- or 'H-labeled L-cystine, L-asparagine, L-histidine, quickly as possible, the hepatic artery and the portal vein were L-glutamine, and 2-(methylamino)-isobutyric acid were purchased severed and the superior vena cava was clamped. At this time, the from New England Nuclear Corp. Carrier L-cysteine plus 5 to 10 mm perfusion was accelerated to 11 ml/min and continued until about 150 dithiothreitol served to produce ['4C]cysteine from ['4C]cystine in the ml of S-77 medium had been used. Following this blanching period, actual media. The unlabeled amino acids were purchased from Sigma Swim's S-77 medium containing 150 units/ml of collagenase was Chemical Co., Calbiochem-Behring Corp., or synthesized in our labperfused through the liver at a flow rate of 7 ml/min. The number of oratory by previously cited procedures (13). Culturing media were milliliters of the collagenase solution used equalled the body weight obtained from Grand Island Biologicals. The highly purified insulin in grams. During the entire perfusion period, the liver was kept moist and glucagon were the generous gifts of Ldy and Co., while the with 0.9% NaCl and at 37OC. After removal of the perfused liver from gentamicin was a donation from Schering Corp. Collagenase was the animal it was placed in either ice-cold Na'-free Krebs-Ringer purchased from either Sigma (No. C-2139) or Worthington Biochembuffer containing 0.1% BSA (cells for immediate study in suspension) ical Corp. (type 11). or Swim's S-77 medium (cells for culture), and the cells were dispersed by gentle agitation. The connective tissue was removed from the cells RESULTS AND DISCUSSION by filtering the suspension through 75 pm mesh nylon cloth. In the Na'-free Krebs-Ringer bicarbonate buffer, 25 mm choline bicarbon- Lack of Inhibition of Glutamine Uptake by MeAIB, and of ate, and 118 mm choline chloride were substituted for the correspond- Cysteine Uptake by Glutamine-Table I shows that MeAIB, ing Na' salts. The cells were then washed four times in one of the which we have convinced ourselves is a model substrate for two media indicated above, selected according to whether they were System A, fails to inhibit significantly the uptake of L-glutato be cultured or not. Centrifugations were at 4 C for 2 min at 50 X mine tested at 0.1 or 2.0 m. The 1-min uptake of L-glutamine, g. Cell viability was typically about 90% by permeability to trypan most of it Na'-dependent, is the highest for any amino acid blue. Cells to be used for culturing were diluted to 0.9 X 10' cells/ml in tested in the hepatocyte (compare with Table 11, Ref. 12). Fig. Waymouths medium (MB752/1) containing 0.2% BSA, 5 pg/ml of 1 shows that glutamine inhibits MeAIB uptake (Kt = 12.1 * sodium oleate, 0.41 mm L-alanine, 0.53 mm L-serine, 62.5 pg/d of 2.2 mm), but in a manner closely approaching the noncompetpenicillin, 5.8 pg/ml of streptomycin, 28.4 pg/ml of gentamicin, and itive. This result is consistent with the conclusion to be drawn 600 microunits/ml insulin. This medium was replaced after 4 h of from Table I, that glutamine is not transported by System A. culturing with the same medium lacking the insulin. Cells were plated Our results differ from those of Joseph et al. (16) who found by applying 3 ml of the above suspension to plastic dishes 60 mm in diameter (Lux Scientific Corp.). Each culture dish had been coated glutamine uptake about half Na'-independent and not signifpreviously with collagen by adding 1.8 ml of a solution containing 50 icantly faster than alanine or serine uptake. These authors pg of collagen/ml (either rat tail collagen isolated by the method of reported competitive inhibition for transport between alanine Pariza et al. (18) or Sigma acid-soluble type I11 collagen, sterilized by and serine, whereas glutamine showed noncompetitive inhifiltration). After 12 to 18 h, the solution was aspirated from the dish. bition of the total uptake of these amino acids (16). This procedure leaves a layer of collagen on the bottom of the dish The uptake of a second system-specific amino acid, L-cysproviding a matrix to which the cells can attach. The cells to be used in the suspension experiments were diluted to about 20 X IOe cells/ml teine at 0.1 mm, was not significantly inhibited by glutamine in Na'-free Krebs-Ringer bicarbonate and left on ice until use. at concentrations up to 25 mm; for example the rates in the Transport Experiments-For the transport experiments with cell absence or presence of 25 ~ lglutamine l ~ were 96.1 k 6.3 and suspensions, amino acid uptake was initiated by adding 0.2 ml of the nmol of L-cysteine.g ICW.min , respectively. cell suspension to 2 ml of Krebs-Ringer bicarbonate buffer (ph 7.4 This finding excludes glutamine uptake by a second Na+and 37OC) containing 0.1% BSA and the labeled amino acid. Bicardependent system, namely ASC, K, for cysteine uptake by bonate-buffered solutions were maintained at ph 7.4 by aeration with 95802, 5% CO,. After the selected interval, the assay was stopped by System ASC being mm. Because no third Na+pouring 10 ml of ice-cold Na'-free Krebs-Ringer bicarbonate buffer dependent transport system for neutral a-amino acids has containing 0.2% sucrose into the incubation flasks. The mixture was been characterized for the hepatocyte, we adopted provisionquickly transferred to conical centrifuge tubes and the cells were ally the view that the rapid Na'-dependent uptake of glutapelleted by centrifugation at 4 C for 2 to 3 min at 2000 X g. The mine occurs by a heretofore unidentified transport system. On supernatant solutions were decanted from the cell pellets and the the basis of the characterization provided by this paper and residual fluid removed from the pellets and the adjacent walls of the tubes by touching the surfaces with strips of fiter paper. The wet cell pellets were then weighed and extracted with 1 ml of 5% sulfosalicylic TABLE I acid. Aliquots of the cell extracts and supernatant solutions were Failure of MeAZB to inhibit L-glutamine uptake assayed for radioactivity and for sucrose (19). The pellet residues L-Glutamine uptake was tested for 1 min at 37 C using freshly after extraction were taken to dryness by heating for 18 h at 100OC. isolated hepatocytes in suspension. The osmotic effects of the inhib- The total water space was taken as the difference in weight between itor were compensated by the addition of choline chloride. The results the fresh cell pellet and the dry pellets obtained after extraction, are the averages f S.D. of three determinations while the extracellular water was estimated as the sucrose space of the fresh cell pellet. All data obtained with cells in suspension are [L-Glutamine] [MeAIB] Cation II expressed in nanomoles of test amino acid per g of intracellular water (Icw) per unit time. Variance is expressed mm by the standard deviation. mm nmol.g 0.1 None 328 Na' * Na' 346 e 17 'L The abbreviations used are: BSA, bovine serum albumin; MeAla, 0.1 Choline None 63 * 6 N-methyl-L-alanine; MeAIB, 2-(methylamino)-isobutyric acid; BCH, 2.0 None 3030 Na' f 54 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid, the b(+) isomer; Na' f 16 ICW, intracellular water; Hepes, 4-(2-hydroxyethyl)-2-piperazineeth- 2.0 None Choline 1080 f 83 anesulfonic acid. Added Neutral Amino Acid Transport System in Rat Liver 4013 for the reasons cited in the introduction, we will refer to it as System N. Specificity as to the Alkali-Ion Co-substrate-System A in the Ehrlich ascites tumor cell tolerates Li'-for-Na' substitution, whereas little or no tolerance of Li' was seen for System ASC in that cell, in the pigeon erythrocyte or the rabbit reticulocyte (3, 10). In contrast, Edmondson et al. (15) found a reversed situation in the rat hepatocyte, leading them to propose that all Li'-stimulated uptake of test amino acids by that cell can be attributed to System ASC. We test the completeness of this reversal in Li' tolerance in a forthcoming paper.' This behavior suggested to us that the ability of Li' to sustain the uptake of glutamine might serve also as a differentiating characteristic for the proposed System N. Table I1 shows that the transport rate for 1 min in the presence of 118 mm Li' was about as great as that in Na'. The apparent distribution ratio for the radioactivity can be easily derived by dividing the uptake in nanomoles. g ICW (PM) by the external concentration (100 PM). These calculations suggest a distribution ratio of between 2 and 3 for the 1-min uptake of [ C]glutamine. The thermodynamic ratio would require chemical analysis of cellular glutamine, as well as its assignment to intracellular compartments, and presumably determination of transmembrane potentials. Note, however, that I P 30.5 mm IO [L-Glutamine], mm FIG. 1. A Dixon plot of L-glutamine inhibition of MeAIB transport. The I-min uptake of MeAIB at 0.1, 0.3, and 0.5 mm was tested in freshly isolated hepatocytes. The results represent the Na'- dependent component of MeAIB uptake only and are the averages of triplicate determinations. The Na+-independent rates which have been subtracted for 0.1, 0.3, and 0.5 mm glutamine uptake were 8.8 f 0.3, 24.1 f 2.1, and 38.1 f 1.1, respectively, i.e. an essentially linear function of concentration. The effects of the added glutamine were compensated by isoosmotic amounts of choline chloride in the corresponding controls. Joseph et al. (16) suggest that under the experimental conditions of Table 11, namely a glutamine concentration of less than 0.5 mm, no significant glutamine catabolism should occur. The demonstrated role of amino acid structure in determining Li' tolerance (22) causes us to hesitate to identify the low tolerance for Li' substitution shown by alanine uptake by System A as a characteristic property of that system in the hepatocyte (12). Note in Table I1 that N-methylalanine (MeAla) does not inhibit glutamine uptake. An unexpectedly high K, for MeAIB in the hepatocyte might have caused us to overlook a weak inhibition of glutamine uptake. In that MeAla is generally more reactive than MeAIB as a transport inhibitor and that alanine inhibits glutamine uptake (see below in Table IV), its inertness reinforces the conclusion that the N-methyl group is unacceptable to the new system. Also note that the Li'- stimulated uptake of glutamine escapes inhibition by MeAla. Effect of External ph-in differentiating Systems A and ASC, the greater sensitivity of System A to increases of [ H+] was valuable both in the Ehrlich cell (3) and in isolated rat hepatocytes (12). Therefore we compared the uptake rates of g glutamine, L-cysteine, MeAIB, and amino-endo(f)2-aminobicyclo(2,2,l)heptane-2-carboxylic acid (BCH) at several ph values between 6.0 and 8.5 (Fig. 2). MeAIB and glutamine showed similarly high sensitivities, whereas uptake of cysteine and BCH were not significantly suppressed by lowering the ph to 6.0. The increased activity of System L corresponds to our experience with the Ehrlich cell (23). Sensitivity to N-Ethylmaleimide-Transport is one of the membrane functions known to depend on membrane-associated sulfhydryl groups. Czech et al. (24) have used this property to contrast the glucose transport system in red blood cells with that in the adipocyte. Table I11 shows that N-ethylmal- P f I I I I I I I 1 TABLE I1 Ability of System N to accept Li'-for-Na' substitution All assays were buffered with Krebs-Ringer solutions containing 25 mm choline bicarbonate, ph 7.4, while the chlorides of Na', Li', or choline were added at 118 mm. Control assays contained 12.5 mm choline chloride to compensate for the osmotic effects of the 25 mm MeAla. The concentration of the L-glutamine was 0.1 mm. The results are the averages f S.D. of three determinations. Inhibitor Cation L-Glutamine uptake + nrnol.g icw.min None 275 Na' f 8 Li' 281 f 3 Choline 20.3 f 0.8 MeAla, 25 mm Na' 290 f 18 Li 290 f6 Choline 22.3 f External ph FIG. 2. The relation between ph of the uptake medium and the uptake by Systems A, ASC, N, and L as measured by MeAIB, L-cysteine, L-glutamine, and BCH, respectively. Hepatocytes in primary culture were assayed at varying ph values between 6.0 and 8.5. The buffer used throughout these experiments was 25 mm Hepes. The data presented for M
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