- Email: [email protected]
Effect of metabolic inhibitors on the respiration of earle strain L cells
506
LE’ITERS
TO
THE
EDITORS
was observed for neutral red. Enzymatic tests on the electrolysis product with alcohol dehydrogenase and lactic dehydrogenase systems showed the reduction compound of DPN to be totally inactive for these enzyme-catalyzed reactions. Previous studies (6) showed that the electrolytic reduction of DPN on the mercury electrode is an irreversible process, since the polarographic El/z is far more negative than the reversible redox potential. Observations made here indicate that the irreversible electrolytic reduction of DPN results in the formation of a compound very similar to but not identical with the enzymatically or chemically (hydrosulfite) reduced DPNH. REFERENCES 1. MATHEWS, M. B., J. B&Z. Chem. 176, 229 (1948). 2. MATHEWS, M. B., AND CONN, E. E., J. Am. Chem. Sot. 76, 5428 (1953). 3. WARBURO, O., CHRISTIAN, W., AND GRIESE, A., Biochem. 2. 262, 157 (1935). 4. SWALLOW, A. J., Biochem. J., 64, 253 (1953). 5. STEIN, G., AND SWALLOW, A. J., Nature 173, 937 (1954). 6. KE, B., Biochim. et Biophys. A&, in press. 7. FISHER, H. F., CONN, E. E., VENNESLAND, B., AND WESTHEIMER, F. H., J. Biol. Chem. 202, 689 (1953). 8. BURTON, R. M., AND SAN PIETRO, A., Arch. Biochem. and Biophys. 48, 184
(1954). 9. MARKHAM, R., AND SMITH, J. D., Biochem. J. 46, 294 (1949). 10. RACKER, E., J. Biol. Chem. 164, 313 (1950). C. F. Kettering Foundation, Yellow Springs, Ohio Received October 19, 1966
BACON
KE
Effect of Metabolic Inhibitors on the Respiration of Earle Strain L Cells1 Studies carried out with microorganisms, plants, and animals have ser,ved to point out the nature of the action of a number of cell poisons on the metabolic pathways in these biological systems. However, a search of the literature has revealed that similar studies, generally, have not been extended to the effect of these metabolic inhibitors on the metabolism of mammalian cells cultured in vitro. The possibilities of studying mammalian cell metabolism in culture are indicated in the present investigation of the effects of the inhibitors 2,4-dinitrophenol, sodium azide, and iodoacetate on the respiratory activity of Earle strain L mouse fibroblasts (1). The cells were grown directly on glass at 37°C. in T60 flasks in an ascitic fluid medium (2) consisting of 40% human ascitic fluid, 40% mixture 199, and 2Oye 1 Supported in part by grants from the United States Public Health National Institutes of Health, and the Research Board of the University fornia.
Service, of Cali-
LETTERS
TO
THE
EDITORS
507
Tyrode solution. Prior to the experiment, the cells were removed from the glass surface by agitation with 0.25% trypsin, washed twice in Tyrode, and resuspended in solutions of graded concentrations of inhibitor in 40% ascitic fluid medium and 60% physiological saline for respiration measurements. The pH of the final suspensions were usually 7.3 to 7.5. The rate of respiration in culture was found to be quite low, the average oxygen consumption of the controls being of the order of 3 ,~l per hour per million cells. To increase the sensitivity of respiration readings, specially constructed Warburg flasks with a total volume capacity of 2.5 ml. were employed. Five-tenths milliliter quantities of cell suspension (2 X lo6 cells/ml.) were added to the main compartments of the reaction vessels, and 0.05 ml. amounts of 10% KOH and filter paper rolls introduced into the center wells. The flasks were then shaken at. 37°C. and readings were made hourly over a 5-hr. period. The effects of graded concentrations of the inhibitors on oxygen consumption by 1, cells are shown in Fig. 1. Each point represents the mean of duplicate or t,riplicate vessels in experiments repeated at least 3 times, and the range is indicated by vertical bars. An arbitrary value of 1.00 has been assigned to the total oxygen consumption of the control cultures (wit,hout inhibitor) over a 5.hr. period. It is observed in Fig. 1 that under the conditions of these experimentas the inhibit,ors demonstrated marked quantitative differences in their effects on oxygen consumption by 1, ceils. Only in the presence of dinitrophenol at concentrations between 1 X 10e5 and 1 X 10e4 A1 was respiration augmented, equivalent concentrations of azide and iodoacetate being either inhibitory or with little effect. Inhibition of respiration by azide commenced at concentrations much higher t.harr those required for iodoacetate; while azide inhibit,ion did not occur to any extent. in conrrntrations below 5 X lo-” M, significant inhihition by iodoacetate way
FIG.
oxygen spective inhibitor
I. Concentration-action curves for 5-hr. period showing relative total consumption of Earle strain L cells as :L function of concent,rat,ion of reinhibitors. An arbitrary value of 1 has heen assigned to the control (no present).
508
LETTER
TO
THE
EDITORS
already apparent at a concentration of 1 X 10-S M. The likelihood of azide distillation to explain the low sensitivity to this inhibitor was considered. However, its presence mainly in the salt form and observations of continued inhibition with time compared to the controls did not substantiate this. These experiments indicate that the effects of metabolic poisons on oxygen consumption by mammalian cells cultured in o&o, while not strictly comparable to effects observed in all biological systems, generally show a relationship to inhibitor concentration similar to that observed with tissues from several sources (3,4,5,6), as best exemplified by the characteristic changes in oxygen consumption spectrum with increasing concentrations of 2,4-dinitrophenol. REFERENCES
1. SANFORD, K. Ii., EARLE, W. R., AND LIKELY, G. D., J. N&Z. Cancer Inst. 9, 229 (1943). 2. CAILLEAU, R., AND KIRK, P. L., J. Natl. Cancer Inst. 16, 295 (1954). 3. MACHLIS, L., Am. J. Botany 31, 183 (1944). 4. PEISS, C. N., AND FIELD, J., J. Biol. Chem. 176, 49 (1948). 5. LATIES, G. G., Arch. Biochem. 20, 299 (1949). 6. INGRAHAM, J. L., ROBY, T. O., AND PETERSON, J. H., Arch. Biochem. and Biophys. 46, 215 (1953). BENJAMIN V. SIEGEL* The Virus Laboratory and RELDA
Department of Biochemistry, University of California, Berkeley, California Received December 30, 1966
2 Aided by a Fellowship
from the National
Foundation
CAILLEAU
for Infantile
Paralysis.
LE’ITERS
TO
THE
EDITORS
was observed for neutral red. Enzymatic tests on the electrolysis product with alcohol dehydrogenase and lactic dehydrogenase systems showed the reduction compound of DPN to be totally inactive for these enzyme-catalyzed reactions. Previous studies (6) showed that the electrolytic reduction of DPN on the mercury electrode is an irreversible process, since the polarographic El/z is far more negative than the reversible redox potential. Observations made here indicate that the irreversible electrolytic reduction of DPN results in the formation of a compound very similar to but not identical with the enzymatically or chemically (hydrosulfite) reduced DPNH. REFERENCES 1. MATHEWS, M. B., J. B&Z. Chem. 176, 229 (1948). 2. MATHEWS, M. B., AND CONN, E. E., J. Am. Chem. Sot. 76, 5428 (1953). 3. WARBURO, O., CHRISTIAN, W., AND GRIESE, A., Biochem. 2. 262, 157 (1935). 4. SWALLOW, A. J., Biochem. J., 64, 253 (1953). 5. STEIN, G., AND SWALLOW, A. J., Nature 173, 937 (1954). 6. KE, B., Biochim. et Biophys. A&, in press. 7. FISHER, H. F., CONN, E. E., VENNESLAND, B., AND WESTHEIMER, F. H., J. Biol. Chem. 202, 689 (1953). 8. BURTON, R. M., AND SAN PIETRO, A., Arch. Biochem. and Biophys. 48, 184
(1954). 9. MARKHAM, R., AND SMITH, J. D., Biochem. J. 46, 294 (1949). 10. RACKER, E., J. Biol. Chem. 164, 313 (1950). C. F. Kettering Foundation, Yellow Springs, Ohio Received October 19, 1966
BACON
KE
Effect of Metabolic Inhibitors on the Respiration of Earle Strain L Cells1 Studies carried out with microorganisms, plants, and animals have ser,ved to point out the nature of the action of a number of cell poisons on the metabolic pathways in these biological systems. However, a search of the literature has revealed that similar studies, generally, have not been extended to the effect of these metabolic inhibitors on the metabolism of mammalian cells cultured in vitro. The possibilities of studying mammalian cell metabolism in culture are indicated in the present investigation of the effects of the inhibitors 2,4-dinitrophenol, sodium azide, and iodoacetate on the respiratory activity of Earle strain L mouse fibroblasts (1). The cells were grown directly on glass at 37°C. in T60 flasks in an ascitic fluid medium (2) consisting of 40% human ascitic fluid, 40% mixture 199, and 2Oye 1 Supported in part by grants from the United States Public Health National Institutes of Health, and the Research Board of the University fornia.
Service, of Cali-
LETTERS
TO
THE
EDITORS
507
Tyrode solution. Prior to the experiment, the cells were removed from the glass surface by agitation with 0.25% trypsin, washed twice in Tyrode, and resuspended in solutions of graded concentrations of inhibitor in 40% ascitic fluid medium and 60% physiological saline for respiration measurements. The pH of the final suspensions were usually 7.3 to 7.5. The rate of respiration in culture was found to be quite low, the average oxygen consumption of the controls being of the order of 3 ,~l per hour per million cells. To increase the sensitivity of respiration readings, specially constructed Warburg flasks with a total volume capacity of 2.5 ml. were employed. Five-tenths milliliter quantities of cell suspension (2 X lo6 cells/ml.) were added to the main compartments of the reaction vessels, and 0.05 ml. amounts of 10% KOH and filter paper rolls introduced into the center wells. The flasks were then shaken at. 37°C. and readings were made hourly over a 5-hr. period. The effects of graded concentrations of the inhibitors on oxygen consumption by 1, cells are shown in Fig. 1. Each point represents the mean of duplicate or t,riplicate vessels in experiments repeated at least 3 times, and the range is indicated by vertical bars. An arbitrary value of 1.00 has been assigned to the total oxygen consumption of the control cultures (wit,hout inhibitor) over a 5.hr. period. It is observed in Fig. 1 that under the conditions of these experimentas the inhibit,ors demonstrated marked quantitative differences in their effects on oxygen consumption by 1, ceils. Only in the presence of dinitrophenol at concentrations between 1 X 10e5 and 1 X 10e4 A1 was respiration augmented, equivalent concentrations of azide and iodoacetate being either inhibitory or with little effect. Inhibition of respiration by azide commenced at concentrations much higher t.harr those required for iodoacetate; while azide inhibit,ion did not occur to any extent. in conrrntrations below 5 X lo-” M, significant inhihition by iodoacetate way
FIG.
oxygen spective inhibitor
I. Concentration-action curves for 5-hr. period showing relative total consumption of Earle strain L cells as :L function of concent,rat,ion of reinhibitors. An arbitrary value of 1 has heen assigned to the control (no present).
508
LETTER
TO
THE
EDITORS
already apparent at a concentration of 1 X 10-S M. The likelihood of azide distillation to explain the low sensitivity to this inhibitor was considered. However, its presence mainly in the salt form and observations of continued inhibition with time compared to the controls did not substantiate this. These experiments indicate that the effects of metabolic poisons on oxygen consumption by mammalian cells cultured in o&o, while not strictly comparable to effects observed in all biological systems, generally show a relationship to inhibitor concentration similar to that observed with tissues from several sources (3,4,5,6), as best exemplified by the characteristic changes in oxygen consumption spectrum with increasing concentrations of 2,4-dinitrophenol. REFERENCES
1. SANFORD, K. Ii., EARLE, W. R., AND LIKELY, G. D., J. N&Z. Cancer Inst. 9, 229 (1943). 2. CAILLEAU, R., AND KIRK, P. L., J. Natl. Cancer Inst. 16, 295 (1954). 3. MACHLIS, L., Am. J. Botany 31, 183 (1944). 4. PEISS, C. N., AND FIELD, J., J. Biol. Chem. 176, 49 (1948). 5. LATIES, G. G., Arch. Biochem. 20, 299 (1949). 6. INGRAHAM, J. L., ROBY, T. O., AND PETERSON, J. H., Arch. Biochem. and Biophys. 46, 215 (1953). BENJAMIN V. SIEGEL* The Virus Laboratory and RELDA
Department of Biochemistry, University of California, Berkeley, California Received December 30, 1966
2 Aided by a Fellowship
from the National
Foundation
CAILLEAU
for Infantile
Paralysis.