STUDIES ON THE EXCHANGE OF POTASSIUM BETWEEN TUMOUR CELL AND MEDIUM

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1 STUDIES ON THE EXCHANGE OF POTASSIUM BETWEEN TUMOUR CELL AND MEDIUM. ARTHUR LASNITZKI (From the Biochemical Laboratory of the Cancer Research Institute, University of Berlin, Germany, and the Cancer Research Department, University of Manchester, England) The present paper deals with the manner in which the exchange of potassium between tumour cell and medium is effected, and the means by which it may be controlled, a subject which appears to be of importance in view of the role of potassium in the life processes within the tumour cell. The investigation was suggested by the results of research on the influence of potassium and calcium upon the energy-metabolism, especially the fermentation capacity, of tumours (2-8). METHODS The experiments were performed in vitro, and the general conditions were, as far as possible, the same as those employed in the metabolism experiments mentioned. Tumour Material: Jensen rat sarcoma was used, being particularly suitable as it contains little stroma. The tumours were produced in the usual way, by subcutaneous implantation of minced tumour tissue in adult rats. Treatment of the Tissue: In each experiment a tumour, removed immediately after the animal had been killed, was divided into several portions, the peripheral areas of which were cut, by means of a razor, into a fairly large number of thin slices. These were put, at room temperature, in groups, into Ringer's solution modified by varying the amounts of potassium and calcium. In the earlier experiments the portions of tumour were immersed in the solution and then sliced, the razor being moistened only with the particular solution concerned. In the later experiments the tumour, while being protected against drying, was cut into slices with a dry razor, and slices were put alternately into the various media. Both methods gave similar results. As far as possible the thickness of the slices was limited to that advocated by Warburg (1), but because of the necessity of employing all the available material the use of somewhat thicker slices could not be avoided. For our investigations, however, this should be of no consequence. After the removal of any necrotic tissue, the tumour slices were washed twice by agitating them carefully in 40 or 50 c.c. of the modified Ringer's solution for ten minutes. Then, after each slice had been rinsed rapidly in distilled water, they were transferred to weighing glasses. In the earlier experiments individual groups of slices were transferred to the weighing glasses one after the other, for the most part in the order in which they had been prepared; in the later experiments the slices belonging to the different groups were, as in their preparation, transferred alternately. In both instances it 51

2 514 ARTHUR LASNITZKI was the intention to equalise, as far as possible, the average time during which the slices in the different groups were kept in contact with the mediurn. The length of this time, always amounting to several hours, depended on the total number of slices to be handled, the amount of necrotic tissue to be removed, the assistance available, and other incidental factors. Frequently, small pieces of tumour, after being freed from any necrotic tissue and rinsed rapidly in distilled water, were transferred to weighing glasses without further treatment. These pieces served for estimating the original potassium content of the tissue. In some of these experiments the investigation of the potassium exchange was restricted to one medium only. Media: The solutions in which the tumour slices were suspended were similar to those used in the experiments on the influence of potassium and calcium upon the fermentation of tumours. Their composition is shown in Table I. TABLE I: Composition of Modified Ringer's Solution, Used as Media Without potassium and calcium Without calcium, with normal content of potassium Without calcium, with increased content of potassium Without potassium, with calcium With potassium and calcium NaCI M M M M M KCI M 0.04 M M CaCI M M NaHCO... ~ M ~ Glucose... ~ 0.2% ~ As will be seen, potassium and calcium chlorides are added to the medium in place of an isotonic amount of sodium chloride. Attention may further be drawn to the fact that the media contain a physiological amount of sodium bicarbonate, but no definite amount of free carbonic acid. This condition existed, also, during the preparation period of the fermentation experiments, but not during the manometric measurements, which were carried out after the media had been saturated with about 5 per cent CO 2 in nitrogen. In the fermentation experiments the media attained, therefore, a rather constant physiological PH, but this was not so in the experiments now being considered. Colorimetric estimations showed that the ph was within physiological limits only in the beginning (- 7. to 7.4), since the bicarbonate stock solutions used for the preparation of the media had been saturated with CO 2 Later the pa gradually increased, finally reaching values of 8.4 to 8.6. It is unlikely, however, that this difference should interfere with a comparison between the two series of investigations. The same may be said of the difference in temperature. Estimation of Potassium: The tissue, placed in weighing glasses, was dried at about 100, and the dry weight then estimated. Afterwards the tissue was ashed by dissolving it in fuming nitric acid, pouring the solution into a micro-kjeldahl flask, and heating with the addition of more fuming nitric acid and of hydrogen peroxide. Finally the ash residue was dissolved

3 EXCHANGE OF POTASSIUM BETWEEN TUMOUR CELL AND MEDIUM 515 in distilled water, and the solution brought to a volume of 25 c.c, Of this solution 10 c.c. were pipetted into each of two centrifuge tubes. (In some cases the solution was, as a whole, transferred into a centrifuge tube, and brought to a volume of 10 c.c.) The estimation of potassium was carried out according to the method of Kramer and Tisdall. After removal of ammonia, the potassium was precipitated by addition of 20 c.c. of the cobaltinitrite reagent to each 10 C.c. of ash solution. The precipitate was then separated by centrifuging, and washed five times with distilled water. The oxidation was carried out in two stages: (1) preliminary oxidation by measured volumes of 0.02 M KMn0 4 solution, and (2) final titration with 0.01 M KMn0 4 solution, both being standardised against 0.01 M oxalic acid. The agreement between the two single estimations was, on the whole, satisfactory. The total amount of potassium was calculated according to the linear equation: K = 2.5 (m V-a) (or, respectively, with 1.0 as factor) in which K is the quantity of potassium in mg., V the (average) volume of used KMnO. solution in c.c. (considered as 0.01 M), while m and a are constants, the value of which had been found by similar estimations carried out, under the same conditions as above, with a series of KCI solutions of known concentrations. (The value of m was 0.069, that of a was ) For small quantities of potassium, however, this equation could not be utilized, the volume of used KMn0 4 solution in this range being less than would be expected. In such cases the determination was performed graphically. From the quantity of potassium thus determined the amount of potassium per 100 mg. dry weight of tissue was finally calculated. RESULTS The individual results obtained in 40 experiments are shown in Table II, the potassium content of the tissue being calculated to 0.05 mg. of potassium per 100 mg. dry weight of tissue. The figures in parentheses indicate the actual amount of tissue (mg. dry weight) employed for the corresponding estimation of potassium. The age of the tumour and the approximate average time during which the slices had been kept in contact with the media are also given. It is to be noted that neither age of tumour nor time of contact, within the given limits, appears to bear any relation to the results. Original Potassium Content of the Tissue: In 27 different experiments the original potassium content of the tissue was estimated. The average value was found to be 1.78 mg. potassium per 100 mg. dry weight. This agrees in order of magnitude with figures obtained by a number of authors working with other tumours, both spontaneous and experimental. As to the variation of individual values, there appears to be a slight tendency for higher values to be more frequent in the later than in the earlier experiments. The standard deviation is In 4 additional experiments, which are not included in the Table, the original potassium content in four or five different parts of the tumour was estimated, without any further test. The difference between the single values

4 516 ARTHUR LASNITZKI and the corresponding mean values averaged about 7 per cent. Similarly in experiments Nos. 18 and 22 the estimation was carried out twice, and here again the agreement was satisfactory. Diffusibility of Tissue Potassium: In 15 experiments the original potassium content of tissue was compared with the potassium content of tissue from the same tumour after suspension in the solution containing neither TARLE II: Potassium Content of Jensen Rat Sarcoma Originally and After Suspension in Different Media (Mg. potassium per 100 mg. dry weight) Ex periment No. Date I 2 Dec. 10, '0 Dec. 18, '0 Jan. 1, '1 4 Jan. 27, '1 5 Nov. 7, '2 6 Nov. 8,'2 7 Nov. 29, '2 8 Dec. 2, '2 9 Dec. 16, '2 10 II Jan. 6, ' Mar.24, ' 12 Apr. 7, ' 1 Jan. 12, '4 14 Jan. 19, '4 15 Jan. 1. '4 16 Feb. 2, '4 17 Feb. 21, '4 18 Feb. 28. '4 19 Mar. 2. '4 20 Mar. 12, '4 21 Mar. 14, '4 22 Mar.20,'4 2 Mar. 26. '4 24 Apr. 9, '4 25 Apr. 12, '4 26 Apr. 25, '4 27 May, '4 28 May 9, '4 29 Nov. 29, '4 0 Nov. 0, '4 1 Dec. 5, '4 2 Dec. 19, '4 Feb. 4, '5 4 Feb. 20, '5 5 Feb. 21, '5 6 Mar. I, '5 7 Mar. 12, '5 8 Mar. 14, '5 9 Mar. 19, '5 40 Apr. 8, '5 Age of the tumor in days after inoculation Tis8ue suspended In Ringer's solution 1----:-----:-----,-----:--, ~i~~~f With contact Ttssue Without Without Without potassium with the non... Without calcium, calcium. potassium, and media suspended potassium with with with calcium (Approximate) and potasaium potassium calcium ( M calcium ( M (0.04 M ( M xci, KCI) KCI) CaCI.) M Hours CaCl.)? (114)? (l05~ ( ( ,00 (20) (91) 0.10 (178) (275) (t96) (156) 0.25 (61) (121) (t42) 0.55 (172) (81) 0.5 (212) (107) (96) (40) 0.25 (106) (41) 0.0 (1) " (70) 0.25 (141) (55) (59) (92) 0.25 (200) * (67) (81) (91) (7) (t9) (4) (4) (11) (80) (7) (57) (62) 28 ~ (4) (5) (16) (60) 0.15 (127) 0.0 (124) 0.0 (86) 0.25 (156) 0.20 (118) 0.5 (67) 0.0 (11) 0.20 (171) 0.45 (4) 0.40 (104) 0,50 (211) 0.0 (162) 0.20 (174) 0.5 (27) 0.75 (77) 0.45 (104) 0.40 (92) 0.40* (108) o.sor (146) 0.45 (9) 0.60 (5) 0.45 (64) 0.0 (107) 0.40 (51) 0.40 (185) 0.60 (46) 0.40 (150) (99) (10) 1.50 (71) (154) (44) (III) (191) (III) 1.15 (210) 1.0 (89) 0.90 (279) 0.5 (196) 0.70 (96) 0.45 (11) (18) (157) (102) (74) * (21) () 0.5 (140) 0.90 (66) 1.60 (52) 1.85t (116) 1.45 (84) 1.60 (66) 0.55 (7) (96) 1.55 (9) (114) 0.95 (204) 0.80 (199) 1.15 (161) 0.90 (61) 1.5 (84) 1.0 (47) 1.5 (127) 1.45 (115) Mean values of two parallel estimations {In parentheses corresponding mean values of the amounts nf t Mean values of three parallel estimations tissue employed. potassium nor calcium. The average potassium content of the non-suspended tissue was here again 1.78 mg. per 100 mg. dry weight, while the corresponding value for the tissue which had been suspended was 0.28 mg. (Fig. 1). The average decrease in potassium content of the tissue was, therefore, about 85 per cent, the loss in potassium being in the majority of experiments between 80 and 90 per cent. From this result it is concluded that at least most of the potassium contained in Jensen rat sarcoma cells is freely diffusible, under the given condi-? ? ~I 1 4 5

5 EXCHANGE OF POTASSIUM BETWEEN TUMOUR CELL AND MEDIUM 517 tions. In order to determine whether the residual potassium 1 could be still further reduced by a prolongation of contact with the medium, in 2 of the experiments (Nos. 1 and 5) part of the tissue was left in the solution for two hours longer than is indicated in the Table. A further decrease of about 0.10 mg. potassium per 100 mg. dry weight was obtained in both cases. Thus it appears that the diffusion of potassium had not been completed within the given time periods, and that the average amount of potassium which can be liberated from the tissue probably exceeds the limit of 85 per cent. The influence of the time factor cannot be deduced, however, by comparison of the results obtained in different experiments. Potassium Content of the Tissue in Relation to that of the Medium: The question now arises as to what extent the potassium content of the tissue can be increased by the addition of potassium to the medium. In 9 experiments the potassium content of tissue after suspension in the solution without potassium and calcium was compared with the potassium content of tissue from the FIG. 1. POTASSIUM CONTENT OF JENSEN SARCOMA AFTER SUSPENSION IN A MEDIUM WITHOUT POTASSIUM AND CALCIUM, COMPARED WITH THE ORIGINAL POTASSIUM CONTENT (AVERAGE OF IS TESTS) In this and the following figures the columns indicating the original potassium content of tissue, and the potassium content of tissue after suspension in media (1) without potassium and calcium, (2) with potassium only, () with calcium only, and (4) with both potassium and calcium, are marked respectively: 0, Na, Na + K, Na +Ca, Na + K + Ca. same tumour after suspension in a similar solution to which a normal amount of potassium ( M KCI) was added. The average potassium content of the tissue per 100 mg. dry weight after suspension in the former medium was 0.27 mg.; after suspension in the latter it was 0.41 mg. (Fig. 2a). Thus the average increase was about 50 per cent. Although the absolute difference appears to be small, its correctness is supported by the fact that, so far as the direction of the result is concerned, all the experiments were in accord. As the potassium content of the medium is increased, the increase in the potassium content of the suspended tissue also becomes more pronounced. In 5 experiments the potassium content of tissue after suspension in the solution without potassium and calcium was compared with the potassium content of tissue from the same tumour after suspension (a) in a similar solution containing potassium in the concentration given above ( M KCI) and (b) in a similar solution containing potassium in a higher concentration (0.04 M 1 In this connection see also other experiments in which the potassium content of tissue after suspension in a potassium-free and calcium-free medium was estimated.

6 518 ARTHUR LASNITZKI KCI). The average potassium content of the tissue per 100 mg. dry weight was 0.0 mg. after suspension in the potassium-free medium, 0.44 mg. after suspension in the medium with a normal potassium content, and 1.6 mg. after suspension in the medium with a high potassium content (Fig. 2b). Thus, while the potassium content of the medium was increased 16 times, the increase in the potassium content of the tissue was, on an average, only about 7.6 times greater. Some variation of this value was observed in individual experiments. Since it appeared possible that the values found for the potassium content of tissue after suspension in the potassium-containing media might be too small, because of washing away of part of the potassium within the tissue while it was being rinsed in distilled water, tests were performed to find out whether the time during which the tissue was in contact with distilled water influenced the result. This was done with the tissue which had been suspended in solution containing M KCI in experiments Nos. 20 and 22, and with the tissue which had been suspended in solution containing 0.04 M a FIG. 2. EFFECT OF THE ADDITION OF POTASSIUM TO THE MEDIUM ON THE POTASSIUM CONTENT OF JENSEN SARCOMA (a) Addition of M KCI (average of 9 tests). (b) Addition of 0.04 M KCI (average of 5 tests). KCI in experiments Nos. 25 and 0. As usual, every tissue slice was rapidly moved to and fro in distilled water, but in experiments Nos. 22 and 0 about one third of the slices were moved only once, a second third were moved 4 times, which corresponds roughly to the ordinary manner of rinsing, and the last third 7 times; while in experiments Nos. 20 and 25 the slices were moved once and 4 times only. It was found that the differences in the potassium content of the tissue, separated in this way, were only slight, if noticeable at all, and were of such a nature that no influence of the kind mentioned could be detected." (It is for this reason that only the mean values obtained from the two or three parallel estimations are given in the Table.) Thus we may assume that the potassium content as estimated represents in each case the true potassium content of the tissue under the given conditions. An approximate calculation was also made of the surplus of tissue potassium, which is due to, and apparently in equilibrium with, the potassium of the medium, per unit of tissue water. The original water content of the tissue 2 In experiment No., however, part of the tissue which had been suspended in the solution containing 0.04 M KCI was washed thoroughly for about one minute in distilled water, and thereby the potassium content of the tissue was reduced to nearly one half.

7 EXCHANGE OF POTASSIUM BETWEEN TUMOUR CELL AND MEDIUM 519 is about 80 per cent, but that of the suspended tissue, especially in the absence of calcium in the medium, is probably somewhat greater. Assuming that the tissue suspended in the solution with M KCI contains per cent water, we shall find that 1 gm. of tissue water contains, on an average, a surplus of mg. potassium. As the medium contains about 0.10 mg. potassium per 1 gm. of water, the average ratio: Increase of potassium concentration in tissue water Potassium concentration in medium water is, therefore, The accuracy of this ratio depends on the validity of our assumption that the water content of the suspended tissue is per cent. With a water content lower than 8 per cent the ratio will increase above 0.29, while if the water content exceeds 87 per cent it will decrease below It must be remembered, also, that only the free water, not that bound to the cell colloids, can be a suitable solvent for potassium. The ratio thereby will naturally be increased, but to what extent we do not know. In any case it may be concluded, with some reservation, that although the average surplus of tissue potassium, due to the presence of potassium in the medium, is rather small, it is at least twice as great as it would be if its distribution throughout the tissue were a matter of solubility alone. The most likely explanation appears to be that part of the potassium concerned is adsorbed by certain structural elements of the tumour cell. Needless to say, the remaining part, which is in true solution, will not be contained in the cells alone, but also, probably to a slight extent, in the spaces between the cells. Our conception is further supported by the fact that the surplus of tissue potassium did not increase 16 times, but, on an average, only about one half that figure, when the medium contained 0.04 M instead of M KCI. While, under this condition, that part of the surplus which is in true solution will probably become 16 times greater, the amount adsorbed must increase less than that. The extent of this latter increase it is difficult to determine, but a simple numerical consideration shows that it cannot be great. Finally, it is quite possible that it is not the surplus but the total amount of tissue potassium which, in reality, is in equilibrium with the potassium of the medium, seeing that a true equilibrium in the potassium-free medium may possibly correspond with a complete absence of potassium in the tissue. Special investigations will be necessary to ascertain this possibility, which would, obviously, favour our conception even more than the result here recorded. Potassium Loss of Tissue in Potassium-containing Media: The rather small increase in the potassium content of the tissue on the addition of M KCI to the medium indicates that, under this condition, the liberation of potassium from the tissue was still considerable. In 10 experiments the potassium content of tissue after suspension in the solution containing M KCI was directly compared with the original potassium content of tissue from the same tumour. An average of 0048 mg. potassium per 100 mg. dry weight was here obtained for the suspended tissue, and an average of 1.92 mg. for the non-suspended tissue, both values being somewhat higher than before (Fig. a). Thus the average decrease of the potassium content

8 520 ARTHUR LASNITZKI was 75 per cent, i.e., only a little less than the average loss of potassium observed in tissue after suspension in the potassium-free medium. This fact appears to be of particular interest because a KCl concentration of about M is approximately that contained in media which, normally, are used for investigations on cell metabolism, both respiration and fermentation. (The same KCl concentration was to a large extent utilized in the metabolism experiments already mentioned.) It will be seen that, on an average, three quarters of the amount of potassium present in tumour tissue as taken from the body is not in equilibrium with the amount of potassium contained in such media. The potassium content has, therefore, to be increased if equilibrium is to be obtained. Even if the potassium content of the medium was 16 times higher than normal, however, complete equilibrium could not be attained. In 7 experiments the potassium content of tissue after suspension in the solution containing 0.04 M KCl was compared with the original potassium content of tissue from the same tumour. An average of 1.61 mg. potassium per 100 mg. dry a b FIG.. POTASSIUM CONTENT OF JENSEN SARCOMA AFTER SUSPENSION (a) IN A MEDIUM CONTAIN ING M KCI (AVERAGE OF' 10 'Ii:sTS) AND (b) IN A MEDIUM CONTAINING 0.04 M KCl (AVERAGE OF 7 TESTS), EACH COMPARED WITH ORIGINAL CONTENT weight was here obtained for the tissue which had been suspended (a higher value than before), and an average of 1.80 mg. for the non-suspended tissue (Fig. b). Thus we had still an average loss in potassium of 11 per cent, and in order to obtain complete equilibrium a further increase in the potassium content of the medium appears to be necessary. With certain reservations the figures giving the average 'percentage decrease of the potassium content of tissue which had been suspended in the potassium-free medium, and in the two potassium-containing media, can be utilized for checking our previous findings regarding the increase in the potassium content of tissue by the addition of potassium to the medium. The figures 85 per cent, 75 per cent and 11 per cent indicate that the potassium content increases, approximately, in the proportions 15: 25 and 15:89 respectively. This would mean for the medium with M KCI an average increase from 0.27 to 0.45 mg. potassium per 100 mg. dry weight, while for the medium with 0.04 M KCI the average increase would be 7.4 times higher. The agreement with the results of our direct estimations is quite satisfactory. The Effect of Calcium: As most of the potassium contained in the tumour cell diffuses freely into a medium in which potassium is absent, and the liberation of potassium is not much less if the medium contains potassium in a

9 EXCHANGE OF POTASSIUM BETWEEN TUMOUR CELL AND MEDIUM 521 physiological concentration, it was of interest to ascertain whether this diffusion could be inhibited by the addition of other cations to the medium. Among the cations in question the most important one appears to be calcium. In 10 experiments the potassium content of tissue after suspension in the solution without either potassium or calcium was compared with the potassium content of tissue from the same tumour after suspension in a similar solution to which a certain amount of calcium ( M CaCI 2 ) was added. On an average, the tissue which had been suspended in the control medium contained 0.1 mg. potassium per 100 mg. dry weight, while with the addition of calcium it contained 0.55 mg. (Fig. 4a). Thus it is evident that the presence of calcium in the medium caused a definite increase in the potassium content of the tissue, the average increase being about 80 per cent. The absolute difference is not great, but its correctness is again supported by the fact that the result of each individual experiment was in the same direction. The effect of calcium generally became more pronounced, both absolutely and relatively, if potassium was present in the medium. In 8 experiments a b FIG. 4. EFFECT OF THE ADDITION OF CALCIl;M ( M CaCl.) TO THE MEDIUM ON THE POTASSIUM CONTENT OF JENSEN SARCOMA (a) Medium without potassium (average of 10 tests). (b) Medium containing M KCl (average of 8 tests). the potassium content of tissue after suspension in the solution containing M KCI was compared with the potassium content of tissue from the same tumour after suspension in a similar solution containing, in addition, M CaCI 2 An average of 0.4 mg. potassium per 100 mg. dry weight was found for the tissue which had been suspended in the control medium, and an average of 1.18 mg. for the tissue which had been suspended in the calcium-containing medium (F:ig. 4b). Thus the average increase in the potassium content of the tissue caused by the presence of calcium in the medium was about 175 per cent. It is to be noted that the average time of contact with the media was approximately the same in this and the former group of experiments (three and a half hours). From these results it is concluded that calcium is capable of inhibiting the diffusion of potassium from the tumour cell into the surrounding medium. There does not seem to be any other possible explanation." The influence B In this connection it is interesting to note that the tissue suspended in the two calciumcontaining media appeared to be more compact and less transparent than that suspended in the calcium-free media, a difference which may well give indications regarding the explanation of the calcium etlect.

10 522 ARTHUR LASNITZKI of calcium appears to be more effective if the medium contains a small amount of potassium. To a certain extent this may be due to a lessening of the diffusion gradient, but, on the other hand, there is the possibility that the presence of potassium in the medium may, as such, support the effect of calcium. Finally, it is probable that the effect would be similar when diffusion takes place in an opposite direction. Potassium Loss of Tissue in Calcium-containing Media: It is clear that the outward diffusion of potassium was, at the given (physiological) calcium concentration, not completely inhibited, but only delayed. This may be quantitatively demonstrated by a comparison of the potassium content of tissue after suspension in the two calcium-containing media with the original potassium content of tissue from the same tumour. For this comparison there are available 6 experiments in which potassium was absent from the medium, and 11 in which it was present. The tissue contained an average of 0.54 mg. potassium per 100 mg. dry weight after suspension in the potassium-free a b FIG. 5. POTASSIUM CONTENT OF JENSEN SARCOMA (a) IN A MEDIUM CONTAINING M CaCI, (AVERAGE OF 6 TESTS) AND (b) IN A MEDIUM CONTAINING M KCI AND M CaCI,.(AVERAGE OF 11 TESTS), EACH COMPARED WITH THE ORIGINAL CONTENT medium, and 1.09 mg. after suspension in that containing potassium, while an average of 1.78 and 1.64 mg. respectively was obtained for the original potassium content of the tissue (Fig. 5a and b). The average decrease in potassium content of tissue was, therefore, 70 per cent in the case of the potassiumfree medium, and 4 per cent in the case of the potassium-containing medium. Thus the average percentage loss of potassium was reduced to about one half if potassium were present in the medium, the average time of contact being approximately the same for both media (three. and a half hours). The figures giving the average percentage decrease in the potassium content of tissue which had been suspended in the two calcium-containing media, and in the two corresponding control media, can be utilized, again with reservation, for checking the results given in the previous section. A decrease of 85 and 70 per cent respectively when potassium is absent, and a decrease of 75 and 4 per cent respectively when potassium is present, are approximately equivalent to an increase in the potassium content of the tissue by the action of calcium in the proportions of 15:0 and 25:66 respectively. This would mean in the former case an increase from 0.1 to 0.62 mg. potassium per 100 mg. dry weight, and in the latter case an increase from 0.4 to 1.1 mg. The agreement is good in one case; in the other it may be considered as satisfactory in view of the very unequal number of control and test experiments.

11 EXCHANGE OF POTASSIUM BETWEEN TUMOUR CELL AND MEDIUM 52 Combined Effect of Potassium and Calcium: Finally, in order to estimate the combined effect of both cations, we have to compare the potassium content of tissue after suspension in the solution containing M KCI and M CaCI" with the potassium content of tissue from the same tumour after suspension in a similar solution free of potassium and calcium. For this comparison 9 experiments are available. An average of 0.26 mg. potassium per 100 mg. dry weight was obtained for the tissue which had been suspended in the control medium, and an average of 1.14 mg. for the tissue which had been suspended in the medium with both potassium and calcium (Fig. 6). The average increase was, therefore, nearly )/;1 times. Thus the combined effect is clearly greater than the sum of the two single effects exercised by potassium and calcium separately. The relative increase is approximately 2)/;1 times with regard to the absolute as well as the percentage figures. This result again demonstrates that the influence of calcium is more effective if the medium contains a small amount of potassium. FIG. 6. En-EeT OF ADDITION OF BOTH POTASSIUM AND CAl.CIUM ( M KCI, M CaCU TO TilE MEDIUM ON THE POTASSIUM CONTENT OF JENSEN SARCOMA (AVERAGJe or 9 TJeSTS) GENERAL DISCUSSION Potassium Exchange and Energy-metabolism: As was mentioned in the beginning of this paper, the investigations considered were suggested by the results of researches on the influence of potassium and calcium upon the energy-metabolism of the tumour cell, in particular its fermentation capacity (2-8). With Jensen rat sarcoma and Flexner-Jobling rat carcinoma, it had been shown that the anaerobic fermentation depended to some extent upon the presence of potassium and calcium in the medium, the increase caused by the two cations being greater than that caused by a single one. As regards the action of potassium alone, the effect occurred not only if the cation was added to the medium immediately, but also if it was added after the tissue had fermented for a while in the control medium. This, however, was not so in the case of calcium. In connection with our observation concerning the relation of the potassium content of Jensen sarcoma to the concentration of KCI in the medium, it is of interest to note that the effect of potassium upon the intensity of fermentation was, on the whole, not much altered in the concentrations used, which varied in nearly all the relevant experiments from to 0.04 M. This result was obtained with both tumours. Thus it must be concluded

12 524 ARTHUR LASNITZKI that, although the surplus of tissue potassium, due to the presence of potassium in the medium, increases greatly (probably in both tumours), this has little influence on the effect of potassium upon fermentation.' This behaviour cannot, meanwhile, be fully explained, although an explanation may perhaps be found by considering the action of potassium as consisting of two components, one of which furthers and the other inhibits the process of fermentation. The fact that calcium in a potassium-free medium was capable of increasing the fermentation intensity only if it was added immediately, but not if it was added subsequently, may be explained by assuming that the action of calcium is indirect, taking place by an inhibition of the diffusion of potassium from the tumour cell into the medium. In this way calcium acts only as long as an outward diffusion of potassium takes place, but is unable to act when diffusion has practically come to an end. We have seen that this explanation is well supported by the result of our investigations on the effect exercised by calcium upon the potassium content of Jensen sarcoma placed in a potassium-free medium. On the other hand, the fact that the fermentation intensity of tissue was greater in a medium containing both potassium ( M KCI) and calcium ( M CaCl~) than in a medium containing only potassium (as above) " might suggest that, even when potassium was present, calcium had acted in a similar manner as in the absence of potassium, on the supposition that the intracellular potassium was not in equilibrium with that of the medium. In view of the fact that the potassium loss in tissue after suspension in a medium with M KCI was not much less than that in tissue which had been suspended in a potassium-free medium, and that the addition of M CaCI~ to the former medium increased the potassium content of the tissue considerably, our explanation is seemingly also correct in the case of the potassium-containing medium. It appears, however, questionable whether calcium acts solely by inhibiting the outward diffusion of potassium, for it is difficult to understand why a definite increase in the fermentation intensity of tissue, compared with that obtained in a medium with KC1, apparently occurred only if the corresponding increase in tissue potassium took place by means of the inhibitory effect of calcium, but not by means of an increase in the potassium concentration of the medium. In addition, it is possible that calcium acts by compensating the presumed inhibitory component of the action of potassium, but if we compare quantitatively the increase in tissue potassium with that in fermentation intensity we must admit that this compensation will not be complete. Finally, it may be remarked that these considerations probably apply, also, to the action of potassium and calcium exercised upon the respiration of the tumour cell. It was found that both cations were capable of increasing the intensity of respiration, the combined effect of both being clearly greater than that of either. The differences observed between the effects upon respiration 4 It appears unlikely that this conclusion will be essentially altered by the differences in the conditions of the two series of investigations, including the difference resulting from the fact that, in the fermentation experiments concerned, potassium was subsequently added to the medium. "The experiments concerned were carried out with Jensen sarcoma alone, but there is little doubt that the result will apply also to the Flexner-J obling carcinoma.

13 EXCHANGE OF POTASSIUM BETWEEN TUMOUR CELL AND MEDIUM 525 and fermentation appeared to be of a quantitative nature only, except for the fact that potassium was unable to act upon respiration if it were subsequently added to the medium. Even this difference, however, does not influence our conclusions. Relation between Intracellular and Extracellular Potassium in the Body: The fact that the potassium originally present in the tumour cell was, under our conditions in vitro, not in equilibrium with the potassium of a medium containing M KCI prompted an inquiry into the behaviour of the tumour cell under natural conditions. If one assumes that the intracellular potassium is freely diffusible to about the same extent in vitro and in vivo, and that the potassium content of the medium surrounding the tumour cell in vivo is not much greater than that of our medium," it is of importance to find the reason for the high potassium content of tumour tissue as taken from the body. In this connection it is possible that in vivo the conditions as to equilibrium are different from those in vitro, in so far as the tumour cell is capable of binding more potassium, for instance, by a greater adsorptive power.' On the other hand, it may well be that the high potassium content is due to an active concentration of potassium by the cell by expenditure of energy, thus producing and maintaining a non-equilibrium. This capacity might disappear outside the body, so that a quick equilibration takes place if it is not interfered with by the presence of calcium. The question which of these two possibilities will ultimately prove to be correct must be left for the future to answer. Obviously the question is part of a general problem concerning the tumour cell as well as the normal cell. SUMMARY ( 1) The exchange of potassium between tumour cell and medium was studied, at room temperature, with Jensen rat sarcoma as the tumour material and modified Ringer's solution, similar to that utilized in fermentation experiments, as the medium. (2) It was found that most of the potassium originally present in the tumour cell was freely diffusible, the potassium content of the tissue being reduced, on an average, by about 85 per cent after it had been suspended in a medium without potassium and calcium. () The average potassium content of the tissue became about 50 per cent greater if M KCI were added to the former medium. Although the absolute increase was rather small, it indicated that part of the surplus of tissue potassium, due to the presence of potassium in the medium, did not exist in true solution but in a state of adsorption. Compared with the original potassium content of the tissue, there remained an average decrease of 75 6 According to the results of P. K. Smith and A. H. Smith (9) the potassium content of rat's serum is about twice as great as that of our medium, while the value found by Heller and Paul (10) is about five times as great. But even if the potassium content of our medium were increased five times, a rough estimation shows that still about SO to 60 per cent of the intracellular potassium could not be in equilibrium with that of the medium. 7 As far as the difference in temperature is concerned, it appears unlikely that the higher temperature of the body will increase the amount of adsorbed potassium, in view of the fact that the temperature coefficient of adsorption is, as a rule, negative.

14 526 ARTHUR LASNITZKI per cent, indicating that the greater part of the potassium originally present in the tumour cell was not in equilibrium with that of the medium. (4) If the KCI concentration of the medium was increased from to 0.04 M, the surplus of tissue potassium increased, on an average, nearly eight times, a finding which from the point of view of adsorption was to be expected. In the medium with 0.04 M KCI the potassium content of the tissue was, on an average, still about 10 per cent less than its original potassium content. (5) The potassium content of the tissue also increased, if M CaCL were added to a medium containing no potassium. The average increase was about 80 per cent, while the average decrease, compared with the original potassium content, was 70 per cent. (6) If the same amount of CaCI~ were added to a medium containing M KCI, a more pronounced increase in the potassium content of the tissue was generally obtained. The average increase was about 175 per cent. Compared with the original potassium content of the tissue, there was now found an average decrease of about 5 per cent. (7) The effect of calcium on the potassium content of the tissue, in the potassium-free as well as the potassium-containing medium, was probably due to an inhibition of the diffusion of potassium from the tumour cell into the medium. (8) The combined effect of potassium ( M KCI) and calcium on the potassium content of the tissue was approximately 20 times greater than the sum of the effects exercised by potassium and calcium separately. (9) The significance of these results with regard to the influence of potassium and calcium on the energy-metabolism of the tumour cell, in particular its fermentation capacity, is discussed, and the relation in which intracellular and extracellular potassium may find themselves in the body is also considered. Note. The author wishes to express his gratitude to Dr. W. Cramer of London, for supplying strains of the Jensen rat sarcoma, and to Dr. M. Lasnitzki for her valuable assistance. REFERENCES 1. WARBURG, 0.: The Metabolism of Tumours, English Translation by F. Dickens, London, Constable and Co., LASNITZKI, A.: Ztschr. f. Krebsforsch. n. 116, LASNITZKI, A., AND ROSENTHAL, 0.: lliochem. Ztschr. 20i: LASNITZKI, A., AND ROSENTHAL, 0.: Biochern, Ztschr. 262: 20, LASNITZKI, A.: Biochem. Ztschr. 264: 285, LASNITZKI, A.: Protoplasm a 22: 274, LASNITZKI, A., AND ROSENTHAL, 0.: Biochem. Ztschr. 281: 95, LASNITZKI, A.: Biochem. Ztschr. 285: 101, SMITH, P. K., AND SMITH, A. H.: J. BioI. Chern. 107: 67, HELLER, V. G., AND PAUL, H.: ]. BioI. Chern. 105: 655, 194.