Concentration of Taurine, ß-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 ß-alanine.—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 ß- 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 37°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 100°.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 filtrâtes 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.—Taurine, ß-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 ß- 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."—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 100°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 100°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, •y-aminobutyricacid (2), and so on, one périment2 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.—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 37°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.