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Endogenous adenosine triphosphate levels in human amnion cells during application of high hydrostatic pressure
Experimental
54
ENDOGENOUS HUMAN
ADENOSINE AMNION
OF HIGH
TRIPHOSPHATE
CELLS
DURING
HYDROSTATIC
J. V. LANDAU’
Cell Research 29, 51-60 (1963)
LEVELS
IN
APPLICATION PRESSURE1
and R. A. PEABODY
General Medical Research Laboratory, VA Hospital; and Department Sub-Department Oncology, and Department of Biochemistry, Albany Medical College, Albany, N.Y., U.S.A.
of Medicine,
Received February 23, 19623
HIGII
hydrostatic pressure has been utilized as a tool for the definitive separation of the phases of the sol-gel cycle in a variety of organisms [ 121 and more recently in the human amnion cell [8]. The effects of high pressure have been measured on the basis of reversible changes induced in the more labile structural components of the cell, the overall shape of the cell, and “relative viscosity” of the cytoplasm. To date, the effect of high pressure on the metabolic processes of the living cell has received only sparing attention. The role of ATP and related compounds in the contraction of a variety of cell-models has been well documented [3]. In living systems it has been shown that ATP can increase relative gel strength [9, 141, can modify streaming patterns when injected into amoebae [2], and can cause form and movement changes in tissue cells when added to their medium [lo]. The isolation of proteins, which are myosin-like in their response to ATP, from slime mold [ 131 and tissue cells [3] has also served to implicate this compound in cytoplasmic movement. 115th these observations in mind, along with the fact that cytoplasmic gelation has been characterized as an endothermic process [ 121 the first phase of our biochemical studies was limited to measurement of endogenous adenine nucleotides during high pressure treatment. This is a report of such measurements on human amnion cells. In addition, the experiments involved a comparison between the results of such treatment on the rapidly growing continuous culture FL amnion cell and on the relatively slo~v primary culture amnion cell. 1 Supported in part by U.S.P.H.S. Grant CY 2664 from the National 2 Mailing address: Veterans Administration Hospital, Albany, N.Y. 3 Revised version received May 2, 1962. Experimental
Cell Research 29
Cancer Institute.
55
Pressure and endogenous ATP MATERIALS
AND
METHODS
The cells used in these experiments were primary amnion and FL amnion. The primary human amnion was routinely prepared by trypsinization of human amniotic membrane obtained within one hour of delivery. The initial FL amnion culture was supplied by Dr. Jorgen Fogh at the 123rd transfer and has been carried to the 237th in our laboratory. PlLSl”RE LlNE
Fig. 1.-A diagrammatic representation of the experimental technique. The medium used for all our cultures was 0.5 per cent lactalbumin hydrolysate and 0.1 per cent yeast extract in Hanks’ balanced salt solution supplemented with 20 per cent pooled human serum. Cells were grown in bottles for some 5-10 days before removal for experimental use. The primary amnion cells could be removed only with 0.05 per cent trypsin whereas FL cells were readily harvested with either trypsin or 0.02 per cent EDTA. As a matter of convenience, EDTA was routinely used with FL cells. However, several experiments on FL cells were run using trypsin and the results were identical with either agent. Once harvested, the cells were gently centrifuged, washed in their growth medium (less the serum), centrifuged again and resuspended in a small amount of medium without serum. This rather dense suspension of cells was then pipetted into a series of small stainless steel chambers, each of which held a volume of approximately 0.4 ml. A hydrostatic pressure of 10,000 lb/in.2 was applied in a matter of seconds to as many as six chambers simultaneously, and each chamber could be sealed off individually by means of its own valve. At various time intervals during the period of pressure Experimental
Cell Research 29
56
J. V. Landau and R. A. Peabody
Fig. 2.-The dismantled pressure apparatus. (A) The pressure valve. (11) The chamber tube. (C) The chamber with pressure couplings. The chamber is 90 mm long and has an internal diameter of about 2 mm. (I)) The terminal block.
application and following pressure release, the chambers were completely immersed in a dry-ice-cellosolve bath at 60°C for almost instantaneous freezing. Following freezing, the small pressure unit was dismantled and the frozen cell suspension ejected into cold 5 per cent perchloric acid (PCA). A diagrammatic representation of this entire procedure is shown in Fig. 1. The actual valve and chamber are shown in Fig. 2. In order to maintain appropriate controls, each experiment was conducted in the following manner. The suspension of cells was pipetted into six pressure chambers and six control chambers. The control chambers were maintained at atmospheric pressure throughout. As each pressure chamber was frozen it was accompanied by one of the atmospheric control chambers. In this manner it was possible to obtain simultaneous data on both pressurized and non-pressurized specimens, each being a representative sample of the same cell suspension. After the frozen cell suspension had thawed in cold PCA, it was centrifuged and the residue was washed with more cold PCA. The supernatants were then pooled and neutralized with KOH and chilled to remove perchlorate. The PCA-insoluble residue was dissolved in NaOH, and protein determinations were made by the method of Lowry et al. [ll]. ATP, ADP and AMP were measured in protein-free filtrates by a combination of enzymatic and spectrophotometric procedures. ATP was measured by either of two methods. In the first method glucose-6-phosphate was formed from ATP and glucose by the addition of hexokinase. The glucose6-phosphate was converted to 6-phosphogluconic acid with TPN and glucose-6phosphate dehydrogenase. The TPNH formed is equal to the ATP content [5]. In later experiments ATP was measured by the ATP-dependent conversion of 3-phosphoglyceric acid and DPNH to 3-phosphoglyceraldehyde and DPN catalyzed by the coupled action of the enzymes, 3-phosphoglyceric acid kinase and triose phosphate dehydrogenase. The decrease in DPNH concentration is equal to the ATP concentration. Experimental
Cell Research 29
Pressure and endogenous ATP
57
ADP was measured by adding phosphoenolpyruvate and pyruvate kinase and converting the product, pyruvate, to lactic acid with lactic dehydrogenase and DPNH. The decrease in DPNH concentration is equal to the ADP concentration [6]. In each of the methods for ATP and in the method for ADP the changes in pyridine nucleotide concentration was made quantitative by the addition of excess substrate (glucose, 3-phosphoglyceric acid and phosphoenol pyruvate, respectively). AMP was measured in early experiments by converting it to IMP with adenylic acid deaminase. The decrease in light absorption at 265 rnp after the enzyme is added is proportional to the AMP content [4]. In later experiments, AMP was measured in the same sample that was used for ADP estimation by adding adenylate kinase to the reaction cuvette after the decrease in DPNH concentration due to ADP content had ceased. Under these conditions any AMP in the sample is converted to ADP and the further decrease in DPNH concentration is proportional to the AMP content [l]. The changes in reduced pyridine nucleotides in these reactions (TPNH increase, DPNH decrease) were measured at 340 mp. The molar absorption coefficient for TPNH or DPNH was taken as 6.22 x 106. All of the reactions described above were carried out in a volume of I ml at 25°C utilizing enzymes of the highest purity. Most of the analyses were carried out with enzymes and all other reagents supplied in kit form from C. F. Boehringer and Sons, Mannheim, Germany. Some analyses utilized high purity enzymes from the Sigma Chemical Company, St. Louis, Missouri (hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase, lactic dehydrogenase). All measurements were performed with a Cary Model 11 Recording Spectrophotometer with an expanded slidewire attachment (O-0.1, 0.1-0.2 optical density at full scale). The sensitivity of detection with this equipment is 0.15 rnp 1cZ of adenine nucleotide under the conditions specified. RESULTS amniorz-Application of 10,000 lbs/in.2 at 35°C resulted in a marked increase (SO-100 per cent) in the level of ATP over a period of about 15 min. Following the rise, this elevated value remained approximately constant (as long as the pressure was maintained) for 30 min, which was the maximum time period used in these studies. The ATP concentration of the atmospheric FL
Fig. 3.-The change in ATP level with time in FL amnion cells under 10,000 Ib/in.2 hydrostatic pressure at 35°C. Each point represents the average of 6 experiments with the vertical extensions representing the range. Maximum level is achieved between 10 and 15 min.
58
V. Landau and R. A. Peabody
J.
controls ATP/mg at about No AMP
remained constant over the entire 30 min period at about 10 mp M protein. These results are shown in Fig. 3. ADP remained constant 8-9 rnp M/mg protein in both the control and pressurized specimens. was detected in either series. When the pressure experiments vvere
Pig. 4.
Fig. 5.
Fig. 4.-The change in ATP level with time in FL amnion pressure at 2°C. Maximum level is achieved within 5 min.
cells under 10,000 lb/in.2 hydrostatic
Fig. 5.-The change in ATP level upon release of pressure at 2’C and 35’C. The cells had been previously subjected to 10,000 lb/in.2 for 20 min. Note the return to normal level within 30 set at 35%.
j
,PRIMARY,
0
5
AMNlON;35’C 10
15
MINUTES
20
Fig. 6.-The ATP level in primary amnion cells during pressure treatment at 35’C. Each point represents the average of 10 experiments. Note that there is little or no change as a result of pressure application. 0 Atmospheric pressure; 0 10,000 lbs/in2.
conducted at 2°C the rise in ATP level was much more rapid although the maximum level reached remained about the same as at 35°C (Fig. 4). At the lower temperature the maximum level was reached within 5 min. Once again, ADP remained constant at 8-9 m~cM/mg protein and no AMP could be detected in either control or pressure series. After 20 min at 10,000 lb/in.2 and 35°C the pressure was released and a almost instantaneously. return to atmospheric pressure was achieved Within 30 set after release of pressure the ATP levels had returned to control values and remained at this level for the next 10 min (Fig. 5). ADP remained Experimental
Cell Reseurch 29
Pressure and endogenous ATP
59
constant throughout and no AMP could be detected. When the experiments were performed at 2”C, no decline in ATP level occurred either on release of pressure or during the next 10 min (Fig. 5). Primary amnion.-Application of 10,000 lb/in.2 at either 35°C or 2°C resulted in little or no increase in the level of ATP. The maximum increase amounted to about 20 per cent but this occurred in only one of 10 experiments. The remainder fell within a 5-15 per cent increase, which may be considered within experimental error (Fig. 6). Atmospheric levels of ATP were identical with those of FL cells. So AMP was detected.
DISCUSSION
The results reported here point out a distinct biochemical difference between primary and FL amnion cells with respect to their reaction to the application of high hydrostatic pressure. The generation time of the FL cell, under our conditions of culture and harvest, was about 17 hr, whereas the generation time of the primary amnion cell was approximately 30 hr. The increase of ATP in FL cells on application of pressure may reflect a metabolic difference between the FL and primary amnion cell. On the other hand, this ATP increase may reflect a physiologic state of the amnion cell during a specific period in the generation time of the cell. One would then have to consider that at any time of cell harvest the proportion of cells in this particular physiologic state in the rapidly grooving FL culture might be substantially greater than that in the primary culture. Some recent experiments on Arbacia eggs, performed in collaboration with Marsland and Asterita have yielded results quite similar to those reported here. Unfertilized eggs, yielded constant adenine nucleotide values throughout experimental treatment, while fertilized eggs, at 35 min post-insemination, responded to pressure treatment in a manner similar to FL amnion cells. The specific details of these experiments will be reported elsewhere. It is obvious that much further work will have to be done on synchronously dividing forms with a carefully controlled time factor to resolve this question. The increase of ATP on pressure application and the decrease immediately after pressure release is difficult to explain. Neither change is reflected in the ADP level, which remains constant, and no ,4MP is present before or after pressure application. If the ATP is metabolically produced during the pressure treatment it would seem to involve an exothermic, process since it is brought about by an imposed negative AV and abetted by a decrease in temperature (Fig. 4). The inhibition of ATP-ase activity under pressure Experimental
Cell Research 29
60
J. V. Landau
and K. A. Peabody
[7] and the restoration of such activity upon release of pressure should result in ADP as well as ATP changes and not in what amounts to an effective increase in total adenine nucleotide. There remains a consideration that the ATP is present as such, but in some undetectable form, in the FL cell. This would mean that a relatively large amount of ATP (10 mpM/mg protein) would normally be insoluble in 5 per cent PCA and is a rather remote probability. Although the increase in ,4TP occurs during the time period encompassing the solation of the cytoplasmic gel and the decrease corresponds precisely with the period of re-gelation, it must be realized that the data presented here do not necessarily delineate a relationship between the two phenomena. They may well be independent consequences of the pressure treatment. It is possible that experiments on isolated cell fractions may throw some light upon the site and nature of the adenine nucleotide formation. Such experiments are currently being planned. SUMMARY
Endogenous ATP, ADP and AMP were measured in both primary and FL human amnion cells. Measurements mere made during hydrostatic pressure-treatment and during the period following release of pressure. FL amnion cells at 35°C displayed an increase in ATP during pressure application and a rapid return to normal levels upon return to atmospheric pressure. There was no concomitant change in ADP and no AMP was detected. Primary amnion cells displayed little or no increase in ATP on pressure application. At 2°C the rise in ATP in FL cells occurred at a more rapid rate than at 35°C and the lower temperature effectively prevented the return to normal values upon release of pressure. REFERENCES 1. BUCHER, T., Aduances in Enzymol. 14, 1 (1953). Reu. Cytol. 1, 135 (1952). 2. GOIJJACRE, R. J., Intern. H., in Cell, Organism and Milieu, p. 45. D. RUDNIK (ed.). Ronald Press, 3. HOFFMAN-BERLING, New York, 1959. H. M., J. Biol. Chem. 167, 461 (1947). 4. KALCKAR, A., J. Biol. Chem. 182, 779 (1950). 5. KORNBERG, A. and PRICER, W. E., J. Riot. Chem. 193, 481 (1951). 6. KORNBERG, K. J. and BEANDALL, A. J., Arch. Biochem. Biophys. 55, 138 (1955). 7. LAIDLER, J. V., Exptl. Cell Research 23, 538 (1961). 8. LANDAU, J. V., MARSLASD, D. and ZIMMERMAN, A. M., J. Cellular Comp. Physiol. 45, 309 9. LANDAU, (1955). H., Cancer Research 12, 847 (1952). 10. LETTR$, N. J., FARR, A. L. and RANDALL, R. J., J. Biol. Chem. 193, 11. LOWRY, 0. H., ROSEBROUGH, 265 (1951). D. A., Intern. Rev. Cytol. 5, 199 (1956). 12. MARSLAND, J., J. Gen. Physiol. 39, 325 (1956). 13. Ts’o, P. 0. P., BONNER, J., EGGMAN, L. and VINOGRAD, 14. ZIMMERMAN, A. M., LANDAU, J. V. and MARSLAND, D., Exptt. Cell Research 15, 495 (1958).
Experimental
Cell Research 29
54
ENDOGENOUS HUMAN
ADENOSINE AMNION
OF HIGH
TRIPHOSPHATE
CELLS
DURING
HYDROSTATIC
J. V. LANDAU’
Cell Research 29, 51-60 (1963)
LEVELS
IN
APPLICATION PRESSURE1
and R. A. PEABODY
General Medical Research Laboratory, VA Hospital; and Department Sub-Department Oncology, and Department of Biochemistry, Albany Medical College, Albany, N.Y., U.S.A.
of Medicine,
Received February 23, 19623
HIGII
hydrostatic pressure has been utilized as a tool for the definitive separation of the phases of the sol-gel cycle in a variety of organisms [ 121 and more recently in the human amnion cell [8]. The effects of high pressure have been measured on the basis of reversible changes induced in the more labile structural components of the cell, the overall shape of the cell, and “relative viscosity” of the cytoplasm. To date, the effect of high pressure on the metabolic processes of the living cell has received only sparing attention. The role of ATP and related compounds in the contraction of a variety of cell-models has been well documented [3]. In living systems it has been shown that ATP can increase relative gel strength [9, 141, can modify streaming patterns when injected into amoebae [2], and can cause form and movement changes in tissue cells when added to their medium [lo]. The isolation of proteins, which are myosin-like in their response to ATP, from slime mold [ 131 and tissue cells [3] has also served to implicate this compound in cytoplasmic movement. 115th these observations in mind, along with the fact that cytoplasmic gelation has been characterized as an endothermic process [ 121 the first phase of our biochemical studies was limited to measurement of endogenous adenine nucleotides during high pressure treatment. This is a report of such measurements on human amnion cells. In addition, the experiments involved a comparison between the results of such treatment on the rapidly growing continuous culture FL amnion cell and on the relatively slo~v primary culture amnion cell. 1 Supported in part by U.S.P.H.S. Grant CY 2664 from the National 2 Mailing address: Veterans Administration Hospital, Albany, N.Y. 3 Revised version received May 2, 1962. Experimental
Cell Research 29
Cancer Institute.
55
Pressure and endogenous ATP MATERIALS
AND
METHODS
The cells used in these experiments were primary amnion and FL amnion. The primary human amnion was routinely prepared by trypsinization of human amniotic membrane obtained within one hour of delivery. The initial FL amnion culture was supplied by Dr. Jorgen Fogh at the 123rd transfer and has been carried to the 237th in our laboratory. PlLSl”RE LlNE
Fig. 1.-A diagrammatic representation of the experimental technique. The medium used for all our cultures was 0.5 per cent lactalbumin hydrolysate and 0.1 per cent yeast extract in Hanks’ balanced salt solution supplemented with 20 per cent pooled human serum. Cells were grown in bottles for some 5-10 days before removal for experimental use. The primary amnion cells could be removed only with 0.05 per cent trypsin whereas FL cells were readily harvested with either trypsin or 0.02 per cent EDTA. As a matter of convenience, EDTA was routinely used with FL cells. However, several experiments on FL cells were run using trypsin and the results were identical with either agent. Once harvested, the cells were gently centrifuged, washed in their growth medium (less the serum), centrifuged again and resuspended in a small amount of medium without serum. This rather dense suspension of cells was then pipetted into a series of small stainless steel chambers, each of which held a volume of approximately 0.4 ml. A hydrostatic pressure of 10,000 lb/in.2 was applied in a matter of seconds to as many as six chambers simultaneously, and each chamber could be sealed off individually by means of its own valve. At various time intervals during the period of pressure Experimental
Cell Research 29
56
J. V. Landau and R. A. Peabody
Fig. 2.-The dismantled pressure apparatus. (A) The pressure valve. (11) The chamber tube. (C) The chamber with pressure couplings. The chamber is 90 mm long and has an internal diameter of about 2 mm. (I)) The terminal block.
application and following pressure release, the chambers were completely immersed in a dry-ice-cellosolve bath at 60°C for almost instantaneous freezing. Following freezing, the small pressure unit was dismantled and the frozen cell suspension ejected into cold 5 per cent perchloric acid (PCA). A diagrammatic representation of this entire procedure is shown in Fig. 1. The actual valve and chamber are shown in Fig. 2. In order to maintain appropriate controls, each experiment was conducted in the following manner. The suspension of cells was pipetted into six pressure chambers and six control chambers. The control chambers were maintained at atmospheric pressure throughout. As each pressure chamber was frozen it was accompanied by one of the atmospheric control chambers. In this manner it was possible to obtain simultaneous data on both pressurized and non-pressurized specimens, each being a representative sample of the same cell suspension. After the frozen cell suspension had thawed in cold PCA, it was centrifuged and the residue was washed with more cold PCA. The supernatants were then pooled and neutralized with KOH and chilled to remove perchlorate. The PCA-insoluble residue was dissolved in NaOH, and protein determinations were made by the method of Lowry et al. [ll]. ATP, ADP and AMP were measured in protein-free filtrates by a combination of enzymatic and spectrophotometric procedures. ATP was measured by either of two methods. In the first method glucose-6-phosphate was formed from ATP and glucose by the addition of hexokinase. The glucose6-phosphate was converted to 6-phosphogluconic acid with TPN and glucose-6phosphate dehydrogenase. The TPNH formed is equal to the ATP content [5]. In later experiments ATP was measured by the ATP-dependent conversion of 3-phosphoglyceric acid and DPNH to 3-phosphoglyceraldehyde and DPN catalyzed by the coupled action of the enzymes, 3-phosphoglyceric acid kinase and triose phosphate dehydrogenase. The decrease in DPNH concentration is equal to the ATP concentration. Experimental
Cell Research 29
Pressure and endogenous ATP
57
ADP was measured by adding phosphoenolpyruvate and pyruvate kinase and converting the product, pyruvate, to lactic acid with lactic dehydrogenase and DPNH. The decrease in DPNH concentration is equal to the ADP concentration [6]. In each of the methods for ATP and in the method for ADP the changes in pyridine nucleotide concentration was made quantitative by the addition of excess substrate (glucose, 3-phosphoglyceric acid and phosphoenol pyruvate, respectively). AMP was measured in early experiments by converting it to IMP with adenylic acid deaminase. The decrease in light absorption at 265 rnp after the enzyme is added is proportional to the AMP content [4]. In later experiments, AMP was measured in the same sample that was used for ADP estimation by adding adenylate kinase to the reaction cuvette after the decrease in DPNH concentration due to ADP content had ceased. Under these conditions any AMP in the sample is converted to ADP and the further decrease in DPNH concentration is proportional to the AMP content [l]. The changes in reduced pyridine nucleotides in these reactions (TPNH increase, DPNH decrease) were measured at 340 mp. The molar absorption coefficient for TPNH or DPNH was taken as 6.22 x 106. All of the reactions described above were carried out in a volume of I ml at 25°C utilizing enzymes of the highest purity. Most of the analyses were carried out with enzymes and all other reagents supplied in kit form from C. F. Boehringer and Sons, Mannheim, Germany. Some analyses utilized high purity enzymes from the Sigma Chemical Company, St. Louis, Missouri (hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase, lactic dehydrogenase). All measurements were performed with a Cary Model 11 Recording Spectrophotometer with an expanded slidewire attachment (O-0.1, 0.1-0.2 optical density at full scale). The sensitivity of detection with this equipment is 0.15 rnp 1cZ of adenine nucleotide under the conditions specified. RESULTS amniorz-Application of 10,000 lbs/in.2 at 35°C resulted in a marked increase (SO-100 per cent) in the level of ATP over a period of about 15 min. Following the rise, this elevated value remained approximately constant (as long as the pressure was maintained) for 30 min, which was the maximum time period used in these studies. The ATP concentration of the atmospheric FL
Fig. 3.-The change in ATP level with time in FL amnion cells under 10,000 Ib/in.2 hydrostatic pressure at 35°C. Each point represents the average of 6 experiments with the vertical extensions representing the range. Maximum level is achieved between 10 and 15 min.
58
V. Landau and R. A. Peabody
J.
controls ATP/mg at about No AMP
remained constant over the entire 30 min period at about 10 mp M protein. These results are shown in Fig. 3. ADP remained constant 8-9 rnp M/mg protein in both the control and pressurized specimens. was detected in either series. When the pressure experiments vvere
Pig. 4.
Fig. 5.
Fig. 4.-The change in ATP level with time in FL amnion pressure at 2°C. Maximum level is achieved within 5 min.
cells under 10,000 lb/in.2 hydrostatic
Fig. 5.-The change in ATP level upon release of pressure at 2’C and 35’C. The cells had been previously subjected to 10,000 lb/in.2 for 20 min. Note the return to normal level within 30 set at 35%.
j
,PRIMARY,
0
5
AMNlON;35’C 10
15
MINUTES
20
Fig. 6.-The ATP level in primary amnion cells during pressure treatment at 35’C. Each point represents the average of 10 experiments. Note that there is little or no change as a result of pressure application. 0 Atmospheric pressure; 0 10,000 lbs/in2.
conducted at 2°C the rise in ATP level was much more rapid although the maximum level reached remained about the same as at 35°C (Fig. 4). At the lower temperature the maximum level was reached within 5 min. Once again, ADP remained constant at 8-9 m~cM/mg protein and no AMP could be detected in either control or pressure series. After 20 min at 10,000 lb/in.2 and 35°C the pressure was released and a almost instantaneously. return to atmospheric pressure was achieved Within 30 set after release of pressure the ATP levels had returned to control values and remained at this level for the next 10 min (Fig. 5). ADP remained Experimental
Cell Reseurch 29
Pressure and endogenous ATP
59
constant throughout and no AMP could be detected. When the experiments were performed at 2”C, no decline in ATP level occurred either on release of pressure or during the next 10 min (Fig. 5). Primary amnion.-Application of 10,000 lb/in.2 at either 35°C or 2°C resulted in little or no increase in the level of ATP. The maximum increase amounted to about 20 per cent but this occurred in only one of 10 experiments. The remainder fell within a 5-15 per cent increase, which may be considered within experimental error (Fig. 6). Atmospheric levels of ATP were identical with those of FL cells. So AMP was detected.
DISCUSSION
The results reported here point out a distinct biochemical difference between primary and FL amnion cells with respect to their reaction to the application of high hydrostatic pressure. The generation time of the FL cell, under our conditions of culture and harvest, was about 17 hr, whereas the generation time of the primary amnion cell was approximately 30 hr. The increase of ATP in FL cells on application of pressure may reflect a metabolic difference between the FL and primary amnion cell. On the other hand, this ATP increase may reflect a physiologic state of the amnion cell during a specific period in the generation time of the cell. One would then have to consider that at any time of cell harvest the proportion of cells in this particular physiologic state in the rapidly grooving FL culture might be substantially greater than that in the primary culture. Some recent experiments on Arbacia eggs, performed in collaboration with Marsland and Asterita have yielded results quite similar to those reported here. Unfertilized eggs, yielded constant adenine nucleotide values throughout experimental treatment, while fertilized eggs, at 35 min post-insemination, responded to pressure treatment in a manner similar to FL amnion cells. The specific details of these experiments will be reported elsewhere. It is obvious that much further work will have to be done on synchronously dividing forms with a carefully controlled time factor to resolve this question. The increase of ATP on pressure application and the decrease immediately after pressure release is difficult to explain. Neither change is reflected in the ADP level, which remains constant, and no ,4MP is present before or after pressure application. If the ATP is metabolically produced during the pressure treatment it would seem to involve an exothermic, process since it is brought about by an imposed negative AV and abetted by a decrease in temperature (Fig. 4). The inhibition of ATP-ase activity under pressure Experimental
Cell Research 29
60
J. V. Landau
and K. A. Peabody
[7] and the restoration of such activity upon release of pressure should result in ADP as well as ATP changes and not in what amounts to an effective increase in total adenine nucleotide. There remains a consideration that the ATP is present as such, but in some undetectable form, in the FL cell. This would mean that a relatively large amount of ATP (10 mpM/mg protein) would normally be insoluble in 5 per cent PCA and is a rather remote probability. Although the increase in ,4TP occurs during the time period encompassing the solation of the cytoplasmic gel and the decrease corresponds precisely with the period of re-gelation, it must be realized that the data presented here do not necessarily delineate a relationship between the two phenomena. They may well be independent consequences of the pressure treatment. It is possible that experiments on isolated cell fractions may throw some light upon the site and nature of the adenine nucleotide formation. Such experiments are currently being planned. SUMMARY
Endogenous ATP, ADP and AMP were measured in both primary and FL human amnion cells. Measurements mere made during hydrostatic pressure-treatment and during the period following release of pressure. FL amnion cells at 35°C displayed an increase in ATP during pressure application and a rapid return to normal levels upon return to atmospheric pressure. There was no concomitant change in ADP and no AMP was detected. Primary amnion cells displayed little or no increase in ATP on pressure application. At 2°C the rise in ATP in FL cells occurred at a more rapid rate than at 35°C and the lower temperature effectively prevented the return to normal values upon release of pressure. REFERENCES 1. BUCHER, T., Aduances in Enzymol. 14, 1 (1953). Reu. Cytol. 1, 135 (1952). 2. GOIJJACRE, R. J., Intern. H., in Cell, Organism and Milieu, p. 45. D. RUDNIK (ed.). Ronald Press, 3. HOFFMAN-BERLING, New York, 1959. H. M., J. Biol. Chem. 167, 461 (1947). 4. KALCKAR, A., J. Biol. Chem. 182, 779 (1950). 5. KORNBERG, A. and PRICER, W. E., J. Riot. Chem. 193, 481 (1951). 6. KORNBERG, K. J. and BEANDALL, A. J., Arch. Biochem. Biophys. 55, 138 (1955). 7. LAIDLER, J. V., Exptl. Cell Research 23, 538 (1961). 8. LANDAU, J. V., MARSLASD, D. and ZIMMERMAN, A. M., J. Cellular Comp. Physiol. 45, 309 9. LANDAU, (1955). H., Cancer Research 12, 847 (1952). 10. LETTR$, N. J., FARR, A. L. and RANDALL, R. J., J. Biol. Chem. 193, 11. LOWRY, 0. H., ROSEBROUGH, 265 (1951). D. A., Intern. Rev. Cytol. 5, 199 (1956). 12. MARSLAND, J., J. Gen. Physiol. 39, 325 (1956). 13. Ts’o, P. 0. P., BONNER, J., EGGMAN, L. and VINOGRAD, 14. ZIMMERMAN, A. M., LANDAU, J. V. and MARSLAND, D., Exptt. Cell Research 15, 495 (1958).
Experimental
Cell Research 29