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Phosphate liberation by endometrium in the presence of adenosinetriphosphate
PHOSPHATE
LIBERATION BY ENDOMETRIUM OF ADENOSINETRIPHOSPHATE”
IN
THE
PRESENCE
HOWARD W. JONES, JR., M.D., RUTH WADE, M.D., AND BENJAMIN GOLDBERG,PH.D., BALTIMORE, MD. (From
the Department
of Gynecology,
John-s
Hoplcins
University
and Hospital)
TUDIES of an alkaline phosphatase, using glyeerophosphate as a substrate, indicate that this enzyme varies during the human menstrual cyc1e.l It was therefore of interest to investigate other phosphate-releasing enzymes in this respect. The present report deals with the biochemical and histochemical variations of phosphate liberation by the endometrium during the normal menstrual cycle in the presence of adenosinetriphosphate (ATP) . s
Methods
and Materials
For the biochemical assays of phosphate-releasing activity the method of DuBois and Potter was used.2 This measures the rate at which inorganic phosphate is released when a tissue homogenate is added to a solution consisting of 0.45 C.C. of 0.013 M ATP, 0.15 C.C. of 0.041 M CaCl,, and 0.45 C.C. of a Verona1 buffer.3 This solution was diluted to 1.95 C.C. with distilled water. These conditions are such that the inorganic phosphate liberated was directly proportional to the tissue concentration and time of incubation. The assays were made at two levels of tissue concentration rather than in duplicate, to provide a better check of the assay. The inorganic phosphate in the tissue homogenates, in the spontaneous breakdown of ATP, and in the reagents, was determined in parallel control .tubes and subtracted from the experimental values. Heating to 68” C. for 10 minutes destroys the ability of the endometrium to release phosphate. The activity is expressed as milligrams of phosphorus released from ATP per gram of tissue in 15 minutes at pH 7.4. In order to test the method in our laboratory we assayed rat muscle, kidney, and liver homogenates and found that they released 13.3, 10.6, and 8.4’ mg. of phosphorus per gram of tissue, respectively. These values were somewhat lower than those reported by DuBois and Potter2 but corresponded to those given by Marquette and Schweigert.’ The endometrial specimens were obtained by curettage, freed from as much superficial blood as possible by the use of gauze or filter paper, and cooled immediately in a beaker immersed in ice water. Portions of each specimen were fixed in 95 per cent ethyl alcohol for histological examination. If the assay could not be done at once, the tissue was kept chilled between 0 and 5” C. Experiments on duplicate samples of tissue showed that the enzyme activity was not affected by the length of refrigeration time. 9 This finding has also been reported by Marquette and Schweigert for rat liver and muscle.4 Since it has been shown that there is no significant variation in the water content of the endometrium during the menstrual cycle,s wet weights were considered to be satisfactory. A torsion balance was used to weigh the tissues and a 5 per cent homogenate was made with distilled water. Tissues were homogenized as quickly as possible with the homogenizing tubes surrounded by ice water. *Aided
by a grant
from
the
American
Cancer
1118
Society,
Maryland
Division,
Inc.
PHOSPHATE
Volume 64 Number 5
LIBERATION
BY
ENDOMETRIUM
1119
The homogenates were added to the substrate mixtures which had previously been brought to 37” C. in a water bath. One tube contained 0.3 C.C. of 5 per cent homogenate and a second tube, 0.6 C.C. At the end of a 15 minute period, the reaction was stopped by the addition of 0.3 C.C. of 50 per cent trichloroacetic acid to each tube. After centrifugation, an aliquot of the supernatant fluid in each tube was taken for inorganic phosphate determination by the Fiske and Subbarow method.6 Histological sections from the alcohol-fixed material were studied to eliminate abnormal eases and dated, using the technique described by Noyes, Hertig, and Rock.’ Histochemical assays of phosphafe liberation were done by an adaptation of Gomori’s technique,8 applied to frozen sections.g Slides were incubated from 15 minutes to 20 hours in solutions containing 0.013 M ATP, 0.036 M CaCl, and Verona1 buffer at pH 7.4 and 9. Frozen sections and sections of alcoholfixed material were treated with the periodic acid-Schiff’s reagent to attempt a correlation between phosphate liberation activity and glycogen formation. Results A total of sixty-eight patients were studied. Thirty-five appeared to have had normal menstrual cycles. In the accompanying bar graph, the results of the normal cases have been summarized. It would appear from these data that a steady increase in activity takes place during the proliferative phase of l;he cycle. The seven values obtained from the twenty-first to the twenty-third days were consistently higher than those of the previous periods. Although activity was high in some cases after this period, an average of the 24 to 28 day perj.od showed a decrease of about 21 per cent.
I)-T I3 0.8Fig.
l.-Milligrams
of
1.7
phosphorus
per
pram
of
endometrium.
The values for phosphate liberation from normal endometrium ranged from 0.8 to 3.54 mg. phosphorus per gram of tissue. Histochemical studies revealed somewhat different findings in phosphate release at pH 7.4 and 9.
JONES,
WADE,
AND
Am. J. Obst. % Gynec. November. 1952
GOLDBERG
At pH 9 there was high activity throughout the dycle in the blood vessels. In general the stromal cells of the endometrium also showed high activity in all phases of the cycle. However, immediately before menstruation there was some lessening of activity. As can be noted in the photomicrograph, the activity seemed to be confined to the cell membrane. The glandular cells showed high activity in the proliferative phase of the cycle (Figs. 2 and 3) but in the luteal phase phosphate liberation was depressed (Fig. 4). At pH 9 most of the activity seemed to be in the cytoplasm or cell membrane. *
Fig.
Fig.
2.
3.
Fig. 2 (Case 97738).-Phosphate liberation at pH 9.0 : day Blood vessels, stromal and gland cells all show heavy concentration incubation.) Fig. 3 (Case 97738).-Periodic-acid Schiff’s reaction : day Little or no glycogen in stroma or glands.
5 of normal of activity. 5 of
normal
menstrual (Three
cycle. hours’
menstrual
cycle.
Volume 61
Number
5
PHOSPHATE
LIBERATION
BY
ENDOMETRIUM
1121
It was the impression that there was a marked depression of glandular phosphate liberation coincident with the marked accumulation of glandular glycogen. In order to study this further, serial frozen sections assayed alternately for glycogen and phosphate liberation were studied. For this purpose, an endometrium which showed considerable glycogen variation from gland to gland was selected. By this procedure, it was possible to confirm the fact that, when glycogen was in high concentration, there was little phosphate liberation and vice versa (Figs. 6 and 7).
Fig. 4 (Case 97261).-Phosphate Blood vessels and stroma strongly Fig. 5 (Case 97261).-Periodic-acid High glycogen content of gland-cell
Fig.
4.
Fig.
5.
liberation at pH 9.0; day 20 of normal menstrual cycle. positive. Glands negative even after 18 hours’ incubat:ion. Schiff’s reaction; day 20 of normal menstrual cycle. cytoplasm.
JONES,
WADE,
AND
GOLDBERG
Am. J. Obst. & Gym. November. 1952
kt pH 7.4, little or no activity was noted in the blood vessel walls. The stroi ma1 and glandular cells showed activity throughout the cycle. For the most ;, there were strong nuclear and somewhat weaker cytoplasmic reaci tions. 52 depression of glandular activity in the luteal phase of the cycle, as I noted at P H 9, was not evident at pH 7.4. Fig.
6.
Fig.
7.
Fig. 6.-Phosphate liberation at pH 9.0. Fig. ‘I.-Periodic-acid Schiff’s reaction. Serial Glands with bgen content from gland to gland. no glycogen (Fig. 7) and vice versa.
section of endometrium high phosphate liberating
with variat ion activity (F ‘ig.
in 5)
Comment In systems as complex as described herein, it is impossible specifically the enzyme or enzymes involved.
to designate
Volume 64 Number 5
PHOSPHATE
LIBERATION
BY
ENDOMETRIUM
Xl23
We have no data which would indicate the number of phosphate ions released or their source. The term adenosinetriphosphatase has been rather loosely used in the literature, but should be reserved for an enzyme which releases only the terminal phosphate from adenosinetriphosphate. These studies may or may not be measuring a true adenosinetriphosphatase. It is unfortunate that at pH 7.4 the histochemical demonstration of phosphate is beset by difficulties arising from the relative solubility of calcium phosphate. The strong nuclear reaction and the general lack of crispness of ,the sections may be an expression of this difficulty. At pH 9.0 these difficulties are overcome in some measure. The correlation of biochemical work at pH 7.4 and histochemical results at the same pH is open to the difficulties of histochemical localization at pH 7.4. No attempt will be made to correlate biochemical assays at pH 7.4 with the histochemical assays at pH 9.0. Be this as it may, the highest phosphate-releasing activity, as determined by biochemical means, coincides with the time of greatest pregnanediol excretion and highest blood progesterone levels.lo, I1 Studies are now under way to investigate in vitro the influence of progesterone and other steroids on the release of orthophosphate from adenosinetriphosphate in homogenate systems. The histochemical assays at pH 9.0 reveal a remarkable suppression of phosphate-releasing activity in the endometrial glands in the luteal phase of the cycle. The disappearance of glandular activity as the cycle progresses is reminiscent of the situation at pH 9 with glycerophosphate as a substrate.l However, there seems to be an intimate correlation between absence of phosphate release in the presence of ATP and glycogen concentration. The fact that glandular and stromal activities differ in the follicular and luteal phases s-uggests that more than one enzyme is involved. Keilley and MeyenhoP2 have described two types of adenosinetriphosphatase in muscle and this may also occur in the endometrium. It is interesting to speculate on the possible relation between glandular activity at pH 9.0 and glycogen deposition. The usual concept of the intermediate metabolism of carbohydrates does not include a role for adenosinetriphosphatase or apyrase. However, it is conceivable that, in the endometrium at least, there may be competitive phosphate-releasing enzymes such as apyrase and/or adenosinetriphosphatase with hexokinase for ATP. This concept implies that enzymes which release phosphate from ATP must be suppressed to allow hexokinase to proceed with the phosphorylation of glucose for the entry through ,the mutase system to glycogen. It may be further supposed that when the adenosinetriphosphatases and/or apyrases are active, the high energy of adenosinetriphosphate is utilized for purposes other than the phosphorylation of glucose. The utilization of this energy for growth immediately suggests itself. If this concept is demonstrated by furt,her studies, the activity of these ph.osphate-releasing enzymes of the endometrium will have a dominant role in controlling endometrial growth and maturation.
Summary The release of phosphate by endometrium in the presence of adenosinetriphosphate has been studied by homogenate and histochemical techniques in various phases of the menstrual cycle. Homogenate studies indicate that there is a cyclic variation of phosphate release which is great,est in the mid-progestational phase of the cycle. Histochemical studies at pH 9 demonstrate an intimate and reciprocal relation between phosphate release and glycogen deposition.
1124
JONES,
WADE,
AND
GOLDBERG
Am. J. Obst.& Gynec. November.1952
References 1. 2. 3. 4. 5. 6. 7. 8. 9.
Atkinson, W. B., and Ginsberg, 8. B.: Cancer 1: 248, 1948. DuBois, K. P., and Potter, V. R.: J. Biol. Chem. 150: 185, 1943. Michaellis, L.: Biochem. Ztschr. 234: 139, 1939. Marquette, Mona, and Schweigert, B. 8.: Science 111: 660, 1950. Stuermer, V. M., and Stein, R. J.: AM. J. OBST. & GYNEC. 61: 668, 1951. Fiske, C. H., and Subbarow, Y.: J. Biol. Chem. 66: 375, 1925. Noyes,. R.. W., Hertig, A. T., and Rock, J.: Fertil. & Steril. 1: 3, 1950. Gomon, C.: J. Cell. & Comp. Physiol. 17: 71, 1941. Jones, H. W., Goldberg, B., and Wade, Ruth. Polysaccharide Synthesis in Tissue of Starved Rat Liver. In press. 10. Forbes, T. R.: AM. J. OBST. & GYNEO. 60: 180, 1950. 11. Jones, G. E. S., Delfs, E., and Stran, H. M.: J. Clin. Endocrinol. 9: 743, 1949. 12. Keilley, W. W., and Meyenhof: J. Biol. Chem. 176: 591, 1949. 110
MEDICAL ARTS BUILDING
Froze?
LIBERATION BY ENDOMETRIUM OF ADENOSINETRIPHOSPHATE”
IN
THE
PRESENCE
HOWARD W. JONES, JR., M.D., RUTH WADE, M.D., AND BENJAMIN GOLDBERG,PH.D., BALTIMORE, MD. (From
the Department
of Gynecology,
John-s
Hoplcins
University
and Hospital)
TUDIES of an alkaline phosphatase, using glyeerophosphate as a substrate, indicate that this enzyme varies during the human menstrual cyc1e.l It was therefore of interest to investigate other phosphate-releasing enzymes in this respect. The present report deals with the biochemical and histochemical variations of phosphate liberation by the endometrium during the normal menstrual cycle in the presence of adenosinetriphosphate (ATP) . s
Methods
and Materials
For the biochemical assays of phosphate-releasing activity the method of DuBois and Potter was used.2 This measures the rate at which inorganic phosphate is released when a tissue homogenate is added to a solution consisting of 0.45 C.C. of 0.013 M ATP, 0.15 C.C. of 0.041 M CaCl,, and 0.45 C.C. of a Verona1 buffer.3 This solution was diluted to 1.95 C.C. with distilled water. These conditions are such that the inorganic phosphate liberated was directly proportional to the tissue concentration and time of incubation. The assays were made at two levels of tissue concentration rather than in duplicate, to provide a better check of the assay. The inorganic phosphate in the tissue homogenates, in the spontaneous breakdown of ATP, and in the reagents, was determined in parallel control .tubes and subtracted from the experimental values. Heating to 68” C. for 10 minutes destroys the ability of the endometrium to release phosphate. The activity is expressed as milligrams of phosphorus released from ATP per gram of tissue in 15 minutes at pH 7.4. In order to test the method in our laboratory we assayed rat muscle, kidney, and liver homogenates and found that they released 13.3, 10.6, and 8.4’ mg. of phosphorus per gram of tissue, respectively. These values were somewhat lower than those reported by DuBois and Potter2 but corresponded to those given by Marquette and Schweigert.’ The endometrial specimens were obtained by curettage, freed from as much superficial blood as possible by the use of gauze or filter paper, and cooled immediately in a beaker immersed in ice water. Portions of each specimen were fixed in 95 per cent ethyl alcohol for histological examination. If the assay could not be done at once, the tissue was kept chilled between 0 and 5” C. Experiments on duplicate samples of tissue showed that the enzyme activity was not affected by the length of refrigeration time. 9 This finding has also been reported by Marquette and Schweigert for rat liver and muscle.4 Since it has been shown that there is no significant variation in the water content of the endometrium during the menstrual cycle,s wet weights were considered to be satisfactory. A torsion balance was used to weigh the tissues and a 5 per cent homogenate was made with distilled water. Tissues were homogenized as quickly as possible with the homogenizing tubes surrounded by ice water. *Aided
by a grant
from
the
American
Cancer
1118
Society,
Maryland
Division,
Inc.
PHOSPHATE
Volume 64 Number 5
LIBERATION
BY
ENDOMETRIUM
1119
The homogenates were added to the substrate mixtures which had previously been brought to 37” C. in a water bath. One tube contained 0.3 C.C. of 5 per cent homogenate and a second tube, 0.6 C.C. At the end of a 15 minute period, the reaction was stopped by the addition of 0.3 C.C. of 50 per cent trichloroacetic acid to each tube. After centrifugation, an aliquot of the supernatant fluid in each tube was taken for inorganic phosphate determination by the Fiske and Subbarow method.6 Histological sections from the alcohol-fixed material were studied to eliminate abnormal eases and dated, using the technique described by Noyes, Hertig, and Rock.’ Histochemical assays of phosphafe liberation were done by an adaptation of Gomori’s technique,8 applied to frozen sections.g Slides were incubated from 15 minutes to 20 hours in solutions containing 0.013 M ATP, 0.036 M CaCl, and Verona1 buffer at pH 7.4 and 9. Frozen sections and sections of alcoholfixed material were treated with the periodic acid-Schiff’s reagent to attempt a correlation between phosphate liberation activity and glycogen formation. Results A total of sixty-eight patients were studied. Thirty-five appeared to have had normal menstrual cycles. In the accompanying bar graph, the results of the normal cases have been summarized. It would appear from these data that a steady increase in activity takes place during the proliferative phase of l;he cycle. The seven values obtained from the twenty-first to the twenty-third days were consistently higher than those of the previous periods. Although activity was high in some cases after this period, an average of the 24 to 28 day perj.od showed a decrease of about 21 per cent.
I)-T I3 0.8Fig.
l.-Milligrams
of
1.7
phosphorus
per
pram
of
endometrium.
The values for phosphate liberation from normal endometrium ranged from 0.8 to 3.54 mg. phosphorus per gram of tissue. Histochemical studies revealed somewhat different findings in phosphate release at pH 7.4 and 9.
JONES,
WADE,
AND
Am. J. Obst. % Gynec. November. 1952
GOLDBERG
At pH 9 there was high activity throughout the dycle in the blood vessels. In general the stromal cells of the endometrium also showed high activity in all phases of the cycle. However, immediately before menstruation there was some lessening of activity. As can be noted in the photomicrograph, the activity seemed to be confined to the cell membrane. The glandular cells showed high activity in the proliferative phase of the cycle (Figs. 2 and 3) but in the luteal phase phosphate liberation was depressed (Fig. 4). At pH 9 most of the activity seemed to be in the cytoplasm or cell membrane. *
Fig.
Fig.
2.
3.
Fig. 2 (Case 97738).-Phosphate liberation at pH 9.0 : day Blood vessels, stromal and gland cells all show heavy concentration incubation.) Fig. 3 (Case 97738).-Periodic-acid Schiff’s reaction : day Little or no glycogen in stroma or glands.
5 of normal of activity. 5 of
normal
menstrual (Three
cycle. hours’
menstrual
cycle.
Volume 61
Number
5
PHOSPHATE
LIBERATION
BY
ENDOMETRIUM
1121
It was the impression that there was a marked depression of glandular phosphate liberation coincident with the marked accumulation of glandular glycogen. In order to study this further, serial frozen sections assayed alternately for glycogen and phosphate liberation were studied. For this purpose, an endometrium which showed considerable glycogen variation from gland to gland was selected. By this procedure, it was possible to confirm the fact that, when glycogen was in high concentration, there was little phosphate liberation and vice versa (Figs. 6 and 7).
Fig. 4 (Case 97261).-Phosphate Blood vessels and stroma strongly Fig. 5 (Case 97261).-Periodic-acid High glycogen content of gland-cell
Fig.
4.
Fig.
5.
liberation at pH 9.0; day 20 of normal menstrual cycle. positive. Glands negative even after 18 hours’ incubat:ion. Schiff’s reaction; day 20 of normal menstrual cycle. cytoplasm.
JONES,
WADE,
AND
GOLDBERG
Am. J. Obst. & Gym. November. 1952
kt pH 7.4, little or no activity was noted in the blood vessel walls. The stroi ma1 and glandular cells showed activity throughout the cycle. For the most ;, there were strong nuclear and somewhat weaker cytoplasmic reaci tions. 52 depression of glandular activity in the luteal phase of the cycle, as I noted at P H 9, was not evident at pH 7.4. Fig.
6.
Fig.
7.
Fig. 6.-Phosphate liberation at pH 9.0. Fig. ‘I.-Periodic-acid Schiff’s reaction. Serial Glands with bgen content from gland to gland. no glycogen (Fig. 7) and vice versa.
section of endometrium high phosphate liberating
with variat ion activity (F ‘ig.
in 5)
Comment In systems as complex as described herein, it is impossible specifically the enzyme or enzymes involved.
to designate
Volume 64 Number 5
PHOSPHATE
LIBERATION
BY
ENDOMETRIUM
Xl23
We have no data which would indicate the number of phosphate ions released or their source. The term adenosinetriphosphatase has been rather loosely used in the literature, but should be reserved for an enzyme which releases only the terminal phosphate from adenosinetriphosphate. These studies may or may not be measuring a true adenosinetriphosphatase. It is unfortunate that at pH 7.4 the histochemical demonstration of phosphate is beset by difficulties arising from the relative solubility of calcium phosphate. The strong nuclear reaction and the general lack of crispness of ,the sections may be an expression of this difficulty. At pH 9.0 these difficulties are overcome in some measure. The correlation of biochemical work at pH 7.4 and histochemical results at the same pH is open to the difficulties of histochemical localization at pH 7.4. No attempt will be made to correlate biochemical assays at pH 7.4 with the histochemical assays at pH 9.0. Be this as it may, the highest phosphate-releasing activity, as determined by biochemical means, coincides with the time of greatest pregnanediol excretion and highest blood progesterone levels.lo, I1 Studies are now under way to investigate in vitro the influence of progesterone and other steroids on the release of orthophosphate from adenosinetriphosphate in homogenate systems. The histochemical assays at pH 9.0 reveal a remarkable suppression of phosphate-releasing activity in the endometrial glands in the luteal phase of the cycle. The disappearance of glandular activity as the cycle progresses is reminiscent of the situation at pH 9 with glycerophosphate as a substrate.l However, there seems to be an intimate correlation between absence of phosphate release in the presence of ATP and glycogen concentration. The fact that glandular and stromal activities differ in the follicular and luteal phases s-uggests that more than one enzyme is involved. Keilley and MeyenhoP2 have described two types of adenosinetriphosphatase in muscle and this may also occur in the endometrium. It is interesting to speculate on the possible relation between glandular activity at pH 9.0 and glycogen deposition. The usual concept of the intermediate metabolism of carbohydrates does not include a role for adenosinetriphosphatase or apyrase. However, it is conceivable that, in the endometrium at least, there may be competitive phosphate-releasing enzymes such as apyrase and/or adenosinetriphosphatase with hexokinase for ATP. This concept implies that enzymes which release phosphate from ATP must be suppressed to allow hexokinase to proceed with the phosphorylation of glucose for the entry through ,the mutase system to glycogen. It may be further supposed that when the adenosinetriphosphatases and/or apyrases are active, the high energy of adenosinetriphosphate is utilized for purposes other than the phosphorylation of glucose. The utilization of this energy for growth immediately suggests itself. If this concept is demonstrated by furt,her studies, the activity of these ph.osphate-releasing enzymes of the endometrium will have a dominant role in controlling endometrial growth and maturation.
Summary The release of phosphate by endometrium in the presence of adenosinetriphosphate has been studied by homogenate and histochemical techniques in various phases of the menstrual cycle. Homogenate studies indicate that there is a cyclic variation of phosphate release which is great,est in the mid-progestational phase of the cycle. Histochemical studies at pH 9 demonstrate an intimate and reciprocal relation between phosphate release and glycogen deposition.
1124
JONES,
WADE,
AND
GOLDBERG
Am. J. Obst.& Gynec. November.1952
References 1. 2. 3. 4. 5. 6. 7. 8. 9.
Atkinson, W. B., and Ginsberg, 8. B.: Cancer 1: 248, 1948. DuBois, K. P., and Potter, V. R.: J. Biol. Chem. 150: 185, 1943. Michaellis, L.: Biochem. Ztschr. 234: 139, 1939. Marquette, Mona, and Schweigert, B. 8.: Science 111: 660, 1950. Stuermer, V. M., and Stein, R. J.: AM. J. OBST. & GYNEC. 61: 668, 1951. Fiske, C. H., and Subbarow, Y.: J. Biol. Chem. 66: 375, 1925. Noyes,. R.. W., Hertig, A. T., and Rock, J.: Fertil. & Steril. 1: 3, 1950. Gomon, C.: J. Cell. & Comp. Physiol. 17: 71, 1941. Jones, H. W., Goldberg, B., and Wade, Ruth. Polysaccharide Synthesis in Tissue of Starved Rat Liver. In press. 10. Forbes, T. R.: AM. J. OBST. & GYNEO. 60: 180, 1950. 11. Jones, G. E. S., Delfs, E., and Stran, H. M.: J. Clin. Endocrinol. 9: 743, 1949. 12. Keilley, W. W., and Meyenhof: J. Biol. Chem. 176: 591, 1949. 110
MEDICAL ARTS BUILDING
Froze?