Concentration of Taurine, ÃŸ-Alanine,And Triiodothyronine By

Concentration of Taurine, ÃŸ-Alanine,andTriiodothyronine by Ascites Carcinoma Cells*

HALVORN. CHRISTENSEN,BENNOHESS, ANDTHOMASR. RIGGS

(Department of Biochemistry and Nutrition, Tufts CollegeMedical School, Boston, Mass.)

In a continuing study of the process of amino would be rather ineffective in an intact animal, acid concentration in this laboratory, with the because during the influx of exogenous a-amino Ehrlich mouse ascites carcinoma cell, we have con acid the limited volume of extracellular fluid would sidered some structures having a different physio soon become swamped with the effluent exchange logical significance than most of the amino acids. substance. Taurine and /3-alanine occur widely in various ani In the present study taurine and /3-alanine were mal fluids and tissues (1, 3, 4, 7), apparently being found to be strongly concentrated by the car localized mainly in the cells rather than in the cinoma cell. Sufficiently large amounts of substances body fluids. In the tissues of numerous marine in reacting with ninhydrin like taurine and /3-alanine vertebrates taurine, in particular, is one of the im were found present in the cell, to make possible portant contributors to the enormous amino acid the uptake of large amounts of amino acids by the levels (10, 11, 13, 15). This substance is also process suggested above. A comparison, however, abundant in the ascites carcinoma cell, a finding of the amount of such substances lost from the which has led us to consider a possible hypothesis cell during the accumulation of glycine, with the as to the mode of amino acid accumulation. amount of glycine accumulated, gave no support We know that the accumulation of substantial for this hypothesis. amounts of one a-amino acid by the cells leads to With regard to the iodine-containing amino the loss of moderate amounts of other free amino acids of the thyroid, the readiness with which they acids from the cells. These losses are by no means enter cells is a very important question, especially as large as the gain which may occur in the amino because of the ability of these substances to inter acid under study, so one cannot conclude that a fere with oxidative phosphorylation (12), pro one-for-one exchange has occurred. Instead, we vided a sufficiently high concentration is reached. have attributed this effect to competition among We have found that during incubation of the tu the amino acids for the transport apparatus. How mor cells with triiodothyronine and thyroxine ever, we have perhaps overlooked j3-amino acids much higher levels of organic iodine are soon such as j3-alanine or taurine as possible "coins of reached in the cellular phase than outside. exchange." In other words, one can visualize that the movement of such a substance in the direction EXPERIMENTAL Taurine and ÃŸ-alanine.â€”Details of collection, of the concentration gradient could be coupled chem ically or energetically with the gain of an a-amino incubation, and sampling of the tumor cells have been given earlier (5). For the study of the ÃŸ- acid against the gradient. In this way the cellular amino acids the medium was Krebs' Ringer-bicar supply of one of these substances might represent the capacity of the cell for capturing amino acids bonate medium, the atmosphere 95 per cent oxy gen and 5 per cent carbon dioxide, the tempera from outside, whenever the extracellular supply is ture 37Â°C. and the incubation time 3 hours. Where increased. Two facts argue against this possibility: First, /3-alanine or taurine were determined the cells were swelling of the cells during the accumulation of suspended in 10 parts of water containing enough acetic acid to bring the pH to about 5, and the amino acids (5) indicates that the process has suspension was held for 5 minutes at 100Â°.The caused an increase in the number of solute mole cules in the cell. Secondly, the process outlined suspending medium was also deproteinized by heating. Measured portions of the filtrÃ¢tes of both * This investigation has been supported in part by a grant cells and suspending fluid were passed through a (C-1268) from the National Cancer Institute, National Insti 100-cm. column containing Dowex-50 (8 per cent tutes of Health, United States Public Health Service. cross-linkage), according to the conditions of Received for publication October 7, 1953. Stein (15). Taurine was eluted with a citrate buf- 124

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1954 American Association for Cancer Research. CHRISTENSENetal.â€”Taurine, ÃŸ-Alanine,Triiodothyronine in Tumor Cells 125 fer, pH 2.5, and appeared within the interval from cumstances with glycine (5). Of the taurine added, 28 to 45 ml., with the peak at 35 ml. In the ÃŸ- 87-101 per cent was recovered after incubation. alanine analyses (6) the elution was with phos Neither free j8-alanine nor carnosine nor an phate at pH 7.4, and the |8-alanine appeared be serine is evident in chromatograms of extracts of tween 42 and 55 ml. the tumor cells.2 An early intention had been to "Non-alpha amino nitrogen."â€”Where the distribution of observe whether /3-alanine was an inhibitor of the the non-alpha amino nitrogen was under study, the cells and concentrating of taurine. Such an inhibition was the suspending fluid were treated with saturated aqueous picric evident from experiments 2 and 4 (Table 1), in acid, 10 volumes and 2 volumes, respectively. a-Amino acids which /3-alanine was present at 10 mM levels. How were determined by the carbon dioxide released at pH 2 in 15 minutes in 10 per cent ninhydrin at 100Â°C.(9). After this ever, a marked swelling of the cells suggested that carbon dioxide had been removed, the pH was raised to 4.7, /3-alanine was also strongly concentrated. In ex- acetate buffer added (8), and the carbon dioxide released in an additional hour at 100Â°C.was collected and measured. We TABLE1 have designated this as the manometric non-alpha amino nitrogen. Under these conditions 1 Mof j3-alaninegave 1.08 Mof UPTAKE OFTAURINE BYASCITESTUMORCELLS carbon dioxide; 1 M of taurine gave 0.18 Mof carbon dioxide. The concentrations are in mM/kg of water. Correction has In 75 minutes 1 Mof 0-alanine yielded 1.18 Mof carbon dioxide. not been made for the taurine initially present in the cellular Van Slyke, Dillon, MacFadyen, and Hamilton (16) recorded phase. Original cell mass 240 mg/flask. Representative experi yields after 6 minutes at pH 4.7 at a 5 per cent ninhydrin ment. level as follows: from /3-alanine, 0.16; from lysine, 1.34; and FINALTAURINIDISTRIBUTION from ethanolamine and glucosamine, traces. They also found that protein hydrolysates continued to yield additional carbon EXPEBI- T\l HIM dioxide after the normal decarboxylation of a-amino acids was IfCNT ADDED Cella Fluid7.47.42320.8Gradient2818516.2Diitri.butionratio4.83.43.31.3 complete. Obviously, the significance of the 103 to 124 msi of 1 10 35.7 carbon dioxide recovered per kilogram of cell water under the 10* conditions used here is quite uncertain; the same may be said 2 25.2 about the 50-90 mu of colorimetrie amino nitrogen found by 3 30 74 4 30* 27.2 application of the ninhydrin procedure of Moore and Stein (14). However, each of these methods was considered to give * 0-alanine added to an initial level of 10 mil. a measure of a broad group of cell constituents including taurine, j3-alanine, â€¢y-aminobutyricacid (2), and so on, one pÃ©riment2 a final /3-alanine concentration of 61 or more of which might possibly participate in an exchange mM was reached in the cells, which would leave with a-amino acids. Therefore, the loss of large amounts of one or more of these from the cell should be reflected in the little in the suspending fluid. Table 2 shows the analyses. The colorimetrie ninhydrin method was applied to uptake of /3-alanine measured by the increase in hot water extracts at pH 5 (as above) or to picric acid extracts the "manometric non-alpha amino nitrogen" dur freed of picric acid by ether extraction in the presence of ing incubation with /3-alanine, compared to cells hydrochloric acid. incubated in the absence of added /3-alanine. Iodine-containing amino acids.â€”Triiodothyronine was added as a 1 mil solution, to 10-100 volumes of suspensions Table 3 shows the shift in the manometric non- of the tumor cells in Krebs" Ringer-phosphate medium. The alpha amino nitrogen and the colorimetrie amino suspensions were then incubated an hour at 37Â°C.in an oxygen nitrogen during the uptake of glycine. While 42 JUM atmosphere. Thyroxin was dissolved in the medium at 0.01- of glycine were being accumulated by 813 mg. of 0.05 mu levels before suspending the tumor cells. After separating and weighing the cells, they were extracted twice cells, the cells lost only 3.5 /ZMof all other com with 6.67 ml. of 15 per cent trichloroacetic acid per gram of pounds giving color with ninhydrin; they sus cells. The insoluble matter was then analyzed for iodine by tained no loss but a slight gain of substances other the method of Zak, Willard, Myers, and Boyle (17), modified than a-amino acids yielding carbon dioxide with according to Astwood.1 The suspending fluid was analyzed ninhydrin. Analyses of the suspending fluid like for total and inorganic iodine and the organic iodine calculated as the difference. wise showed that the loss of these categories by the cells was by no means as great as the gain of gly RESULTS cine. The total amount of material reacting with The freshly collected tumor cells were found to ninhydrin was increased during incubation with contain 18-20 mM of taurine/kg of cell water, glycine by 16 (manometric method) or 28 /J.M. whereas the ascitic plasma contained only a very (colorimetrie method). Even if this new material small amount, of the order of 0.01 mM. Upon in were all generated in the cell, the amount is not cubation with 10 mm and 30 mM taurine the in large enough to support the existence of an ex crease in the taurine concentration in the cells change process. was much greater than that in the fluid (Table 1), Interestingly, the categories of non-alpha amino and gradients of more than 50 mM were obtained, nitrogen distributed themselves between the cells comparable to those obtained under similar cir- and a saline suspending fluid, much in the fashion 1 E. B. Astwood, personal communication. *Schmidt and Cubiles, unpublished observation.

of the a-amino acids, with distribution ratios be free iodide. The much greater solubility and better tween 6 and 11. recovery of triiodothyronine, plus the sensitivity Table 4 shows the results with triiodothyronine. of the concentration process to cyanide (Exp. Sixty-two to 100 per cent of the added organic 11 B), support the view that this amino acid iodine was recovered. The organic iodine of the does not escape the cellular concentration proc cells corresponded initially to 0.00026 mia triiodo ess to which all other amino acids so far studied are thyronine per kg water. After an hour this value subject.

TABLE 2 UPTAKEOF^-ALANINEASMEASUREDBYTHEINCREASEINTHE MANOMETRICNON-ALPHAAMINONITROGEN Concentrations are in mM/kg of water. Each flask contained 5 ml. of cell suspension. The final cell masses were: A, 87ÃŽmg;B, 944 mg; C, 978 mg. Representative experiments. Initial oi 0-alanine nonalpha ratio of analyzed in medium08459a-aminoN21.2 aminoN74.2 aminoN65 excÃ¨sÂ»N1.81.35 AncPhase Cells Susp.fluidCells 1.9717.7 6.7139

Susp.fluidCells 3.0217.0 42.6152 35.978

Susp. fluid 3.69Nonalpha 64.3Excess 57.6Distribution

TABLE 3 EFFECTOFTHE UPTAKEOFGLYCINEONTHEDISTRIBUTIONOFOTHER SUBSTANCESREACTINGWITHNINHYDRIN The results are with a single lot of cells. B represents the cells incubated in Krebs" Ringer-bicarbonate without added glycine; lot C, with glycine added to an initial concentration of 35 mM. The gains and losses shown ore for 813 mg. of fresh cells. Representative experiment.

GLYCINE MANOMETBIC COLOHIMETRIC AMINO N GLTCINE FOUND NON-ALPHA Total Less glycine ADDED IN mu/KO AMINONITROGEN mu/kg mM/kg lili WATEB mu/KO WATER water water ABCFreshcellsIncub. O 3.89 103 49.5 45.6 cells 8.43 06 Susp.fluidIncub. O 0.75 10.46618.3MM3.3*4.9810335.4MM38.4.23869.9M" cellsSusp. 67.2 fluidGained 166 25.5 IM by cells 42.2 7f - 3.5* Gained by fluid -49.2 13.U53.0 - 22.7f44.6 26.5t Â»These values should be â€”42if a stoichiometric exchange had occurred. t These values should be zero if a stoichiometric exchange of other amino compounds for glycine had occurred. t These values should be +49 if a â€¢toichiometricexchange had occurred.

was enormously increased, so that the added organ TABLE 4 ic iodine was 10-25 times as high inside the cells as UPTAKEOFTRIIODOTHYRONINEBY outside, on a water basis. Similar results were ob ASCITESTUMORCELLS tained with thyroxine at similar concentrations Each equilibration was with approximately 6 ml. of cell with distribution ratios of 14.7 to 32, although two suspension containing 0.6 gm. of cells, fresh weight. Concentra factors lead us to regard these results with thy tions are in M/kg water. roxine as less significant than those obtained with FINAL TBIIODOTHTBONINEDISTRIBUTION Distri- triiodothyronine: (a) thyroxine with its low solu EXPERI INITIAL bution bility is very liable to adsorption upon protein MENT LEVEL Cells Fluid ratio particulates3 and may have been carried down on 11A 0.091 0.63 0.029 21.4 the outside of the tumor cells; (Ã¨)considerable de- B 0.091* 0.53 0.055 9.7 C 0.010 0.047 0.018 25.6 iodination occurred with thyroxine, with recoveries 12A 0.100 0.49 0.051 9.7 of 45-57 per cent and concomitant increases in the B 0.050 0.20 0.017 11.6 * KCN, 1.Ã•mM initially present. Aeration was with HCN-free oxygen, *F. L. Hoch, personal communication. so much of the cyanide may nave been swept out as HCN.

SUMMARY chromatographique concernant l'acide pantothenique de l'urine. Bull. Soc. Chim. Biol., 33:1209-13, 1951. Taurine, /3-alanine, and triiodothyronine were 7. DENT, C. E. Behavior of Sixty Amino Acids and Other found to be strongly accumulated by the Ehrlich Ninhydrin Reacting Substances on Phenol-collidine mouse ascites carcinoma cell upon incubation in Filter-Paper Chromatograms, with Notes on the Occur vitro. No indication was obtained that cellular sub rence of Some of Them in Biological Fluids. Biochem. J., stances of the nature of taurine and /3-alanine ex 43:169-80, 1948. 8. HAMILTON,P.B. Gasometric Determination of Glutamine change mole for mole with extracellular amino Amino Acid Carboxyl Nitrogen in Plasma and Tissue acids during amino acid accumulation. These re Filtrates by the Ninhydrin-Carbon Dioxide Method. sults support the view that the normal localization J. Biol. Chem., 168:375-95, 1945. of taurine and /3-alanine in cells containing them is 9. HAMILTON,P.B., and VANSUM., D. D. The Gasometric Determination of Free Amino Acids in Blood Filtrates by due to an active process for concentrating them the Ninhydrin-Carbon Dioxide Method. J. Biol. Chem., there; it is further indicated that triiodothyronine 160:231-50, 1943. 10. HENZE,M. Beitrage zur Muskelchemie der Octopodin. II. may be expected to reach effective levels readily Mitteilung. Ztschr. f. Physiolog. Chem., 43:477-93, 1904. within cells when added to the extracellular phase. 11. KELLT, A. Beobachtungen ueber das Vorkommen von Aetherschwefelsaeuren, von Taurin und Glycin bei REFERENCES niederen Tieren. Beitr. Chem. Physiol. u. Path., 6:377- 1. AWAPARA,J.;LANDUA,A.J.; and FUERST,B. Distribution 83, 1904. of Free Amino Acids and Related Substances in Organs of 12. MARTIUS,C., and HESS, B. The Mode of Action of Thy- the Rat. Biochim. et Biophys. Acta, 6:457-62, 1950. roxine. Arch. Biochem. et Biophys., 33:486-87, 1951. 13. MENDEL,L. B. Ueber das Vorkommen von Taurin in den 2. AWAPARA,J.;LANDUA,A.J.; FUEHST,R.; and SEALEB. Free 7-Aminobutyric Acid in Brain. J. Biol. Chem., 187: Muskeln von Weichtieren. Beitr. Chem. Physiol. u. Path., 85-39, 1950. 6:582, 1904. 14. MOORE, S., and STETN,W. H. Photometric Ninhydrin 3. BOULANGER,P.,and BISERTE,G. Free Amino Acids and Method for Use in the Chromatography of Amino Acids. Peptides of Brain Tissueâ€”Presence of Combined 7-Amino J. Biol. Chem., 176:367-88,1948. butyric Acid. Compt. rend. Soc. de biol., 233:1498-1500, 15. STEIN, W. H. A Chromatographie Investigation of the 1951. Amino Acid Constituents of Normal Urine. J. Biol. Chem., 4. CAMPBELL,P. N., and Work, T. S. Fractionation of the 201:45-58, 1953. Nitrogenous Water-soluble Constituents of Liver. 1. The 16. VANSLYKE,D. D.; DILLON,R. T.; MACFADYEN,D. A.; Isolation of Glycerlphosphorylethanolamine and of and HAMILTON,P.Gasometric Determination of Carboxyl Taurine. Biochem. J., 60:449-54,1962. Groups in Free Amino Acids. J. Biol. Chem., 141:627-69, 5. CHRISTENSEN,H. N., and RÃaos, T. R. Concentrative 1941. Uptake of Amino Acids by the Ehrlich Mouse Ascites 17. ZAK,B.; WILLARD,H.H.; MYERS,G. B.; and BOYLE,A. J. Carcinoma Cell. J. Biol. Chem., 194:57-68, 1952. Chloric Acid Method for Determination of Protein-bound 6. CROKAEHT,R.; MOORE,S.; and BIOWOOD,E. J. Ã‰tude Iodine. Anal. Chem., 24:1345-48,1952.

Halvor N. Christensen, Benno Hess and Thomas R. Riggs

Cancer Res 1954;14:124-127.

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