INFLUENCE OF ESTROGEN ON THE ELECTROLYTE PATTERN OF THE IMMATURE RAT UTERUS*

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1 INFLUENCE OF ESTROGEN ON THE ELECTROLYTE PATTERN OF THE IMMATURE RAT UTERUS* BY N. B. TALBOT OLIVER H. LOWRY AND E. B. ASTWOOD (From the Biological Laboratories Harvard University Cambridge and the Departments of Biological Chemistry and Pediatrics Harvard Medical School Boston) (Received for publication October ) The present paper reports measurements on the electrolyte content of immature rat uteri before and after the administration of a single dose of estrogen. It has been shown previously that estrogen causes a prompt and striking increase in the weight and water content of rat uteri and that these changes are followed by a further gain in weight associated with an increase in mitotic activity and protoplasmic growth (1). It is to be expected that there should be shifts in the electrolytes to correspond with the ebb and flow of water. If this thesis is correct these phenomena should offer an unusual opportunity to study the effect of estzogen on electrolytes and to investigate electrolyte metabolism during normal protoplasmic growth. Furthermore the pattern of the electrolytes gives some information concerning the location of the water which is responsible for the increase in uterine weight. The data obtained show that estrogen causes a significant increase in the sodium chloride and calcium content of the uterus during the period of edema and later during the period of protoplasmic growth an increase in potassium phosphorus and magnesium content. At the period of greatest edema (6 hours after estrogen administration) the electrolyte pattern differs considerably from that of untreated controls. The pattern suggests that the edema is both extracellular and intracellular. At the peak of uterine growth (30 hours after estrogen administration) the * This work was aided in part by a grant from the Rockefeller Foundation administered by Professor F. L. Hisaw. 1

2 2 Estrogen Influence on Electrolytes electrolyte pattern has almost returned to its control configuration. Materials and Methods One hundred and eighteen 21 to 22 day-old immature litter mate rats from an inbred colony were used. The average uterine weight of 21 to 26 day-old rats in our colony is 20 mg. and seldom varies more than 2 mg. The uterine weight response to a single dose of estrogen has been observed frequently and found to be consistent. Rats were killed with chloroform before and at varying intervals after the administration of 0.1 microgram of a-estradiol in 0.1 cc. of sesame oil subcutaneously. The uteri were removed immediately blotted on filter paper dissected free from adjacent tissues and placed in tightly stoppered tubes. The uteri of several rats were usually collected in one tube for analysis but in one series of twenty-four rats the uteri were collected separately and analyzed individua1ly.r In Group A (Table I) a sample of pooled blood was obtained with a dry syringe during life and the serum analyzed. The hearts of the same group were removed at autopsy pooled in a test-tube after being blotted carefully and analyzed. The methods employed will be described in detail by one of us (0. H. L.) elsewhere.2 The fat-free solid weight was obtained according to the directions of Hastings and Eichelberger (2). Chloride was determined by Volhard titration sodium as sodium zinc uranyl acetate and potassium as potassium chloroplatinate gravimetrically. Calcium was precipitated as the oxalate and titrated after conversion to carbonate. Magnesium was precipitated as magnesium ammonium phosphate and the phosphate determined. The total phosphorus was determined colorimetrically with the Fiske and Subbarow (3) reagents. A single sample of about 0.3 gm. of tissue representing ten to twenty uteri was used for the determinations on pooled samples. On this amount of tissue the error of the methods is estimated at f2 per cent for Cl and I? =t3 per cent for Mg 0 to 5 per cent for K and Na and &5 per cent for Ca. 1 Four groups of animals were studied at different times. These are presented as Groups A B C and D in Table I. The measurements on individual rats are shown in Fig Lowry 0. H. unpublished data.

3 TABLE I Observations on Water and Electrolytes of Immature Rat Uteri Hearts and serum before and ajter Administration of Xingle Dose of c+estradiol Period - Material Amount per kilo fat-free tissue Extracellular Hz0 per kilo fat-free tissue* 1IltIWY31- lular Hz0 per kilo ce1lst Control 6 hrs. 30 hrs. Pooled uteri I Hearts Serum Pooled L uteri L 6 L I Hearts Serum Pooled uteri I 6 6 Hearts Serum 4 z 3 - A B A L I B C D A C I B D A LL _- - gm. gm : : t L 14.8 I ~ ~ - m. m. m.eq.?nm m.ep. m.eq. 7n.q. n.q ' E C ' I l.oe mg. mg. mg. gm. gm. m.e*. 28.: : E E : 22.5 i E i 24.E! 4.1: : ! 24.C 1 3.8: t * The extracellular space was estimated by assuming that all the chloride or sodium was extracellular and at a concentration of 115 and 145 milliequivalents per kilo respectively except in Group A in which the concentration was derived from serum data as shown in the text. t The Hz0 per kilo of cells was calculated by subtracting the estimated gm. of extracellular water from the total tissue water and dividing this by the total weight minus the extracellular weight. G 2 g 8 H k.l 39.t 33.E 6.Oi C t C : : : L C 672 7Of : 31.c C i ) - - I - i ) I

4 4 Estrogen Influence on Electrolytes Results Immature Rats. Group A-The data obtained (Table I) show that a single dose of a-estradiol caused definite changes in the 65C "w "w 3oc a s 25c 'p IX IOC HR. 6 HR. 30 HR. FIG. 1. Average electrolyte content of uteri 0 6 and 30 hours after the administration of 0.1 microgram of cu-estradiol. electrolyte pattern of the uteri but not of the hearts or blood serum. All weights are presented on a fat-free basis. The chief features are as follows: (a) 6 hours after the administration of ar-estradiol when the uteri were edematous as evidenced by a

5 .. I Talbot Lowry and Astwood 5 decrease in the total solids per kilo the concentration of sodium had risen significantly and that of chloride slightly whereas the concentrations of potassium and phosphorus had fallen. Although I x XNP l * x... l fq$ *ii l * #. CI TOTAL z IO DRY WEIGHT 6.1== P & CONTRZL WEIGHT 5 IO SD HOURS AFTER INJECTION FIG. 2. Observations on the concentrations of electrolytes per kilo of fat-free tissue in individual uteri before and after the administration of 0.1 microgram of a-estradiol. 0 = milliequivalents of chloride 0 = mix of phosphorus X = milliequivalents of sodium in individual uteri. The fine lines in the upper portion follow the changes in phosphorus (Curve l) sodium (Curve 2) and chloride (Curve 3) respectively. The two curves in the lower portion show the changes in uterine weight reported previously (1). -8 the concentration of potassium per kilo of tissue had fallen the amount of potassium per kilo of total solids had risen as had the ratio of potassium to phosphorus. (b) 30 hours after a-estradiol

6 6 Estrogen Influence on Electrolytes administration the uteri were much larger than control uteri but were no longer edematous. These uteri had essentially the same electrolyte pattern per kilo of tissue as did control uteri. When the average total electrolyte contents of uteri 0 6 and 30 hours after a single dose of estrogen are calculated (Fig. l) it becomes evident that there was a striking increase in the electrolyte content of the 6 and 30 hour samples. The cation columns are probably quite accurately representative of the tissue. The anion columns are less accurate because the valence of the various phosphorus compounds is not known definitely. In Fig. 1 a valence of 1 has been assumed for phosphorus. A represents unmeasured anions (HCOs- etc.). At 6 hours there was a relative and absolute increase in A. The electrolyte content per kilo of individual uteri is shown in Fig. 2. That changes in the electrolyte content of the uteri precede changes in water content is the only obvious interpretation of Fig. 2. More data are necessary to establish that point however. DISCUSSION In this discussion sodium and chloride are considered to be chiefly extracellular elements and potassium magnesium and phosphorus intracellular elements. It is recognized however that any of these elements may be present to some extent in both phases. The changes in the electrolyte pattern observed at 6 hours arc of interest because they probably precede mitotic activity. At that time the average increase in the uterine water content was 67 per cent. The chloride increase (+63 per cent) and the sodium increase (+I16 per cent) were approximately what would be expected if two-thirds of the added water was ultrafiltrate. The increase in potassium and magnesium (f33 per cent) was greater than the increase in solids (f-27 per cent). The phosphorus on the other hand rose less (+12 per cent) than the solids. The absolute increases in the potassium phosphorus and magnesium per uterus without a proportional increase in cell number suggest that there must have been an increase in the cell membrane permeability to these elements. It also seems necessary to consider the possibility of an increase in cell permeability

7 Talbot Lowry and Astwood 7 to sodium and chloride as well. At 30 hours the electrolyte pattern has returned approximately to the control configuration even though the uteri have increased 100 per cent in total weight. In most tissues the extracellular fluid probably resembles a serum ultrafiltrate. If that is so the ratio of sodium to chloride (as derived from the Donnan ratio applied to serum values) in the extracellular tissue phase should approximate A lower ratio 0.95 was observed in the control uteri and a ratio of 1.25 in the 6 hour uteri. These findings suggest that at least in the control uteri some of the chloride was intracellular. Manery and Hastings (4) have described other tissues in which there appears to be some intracellular chloride. If some of the chloride is intracellular chloride cannot be considered an accurate index of the volume of extracellular fluid. On the other hand because the sodium in the uteri appears to be almost exclusively extracellular it should serve as an index of the extracellular fluid volume. The data (Table I) support this thesis when the concentration of sodium and chloride in the ultrafiltrate was calculated by assuming a Donnan ratio of 0.95 (2). They were found to approximate 145 and 115 millicquivalents per kilo respectively. On the basis of these values the extracellular volume was calculated and from that the water per kilo of cells was found by subtracting the estimated gm. of extraccllular water from the total tissue water and dividing that value by the total weight less the extracellular fluid weight. When this was done it was found that the chloride values gave cell water contents considerably below 700 gm. per kilo for the control and 30 hour uteri whereas with a few exceptions the sodium data gave calculated cell water contents that approximated those of the rat hearts and of other striated muscle tissue (2). This is regarded as further evidence that sodium was chiefly extraccllular in these uteri and that it therefore should scrvc as an approximate index of the extracellular fluid content. Fig. 3 shows the average changes in total solids and intracellular and extracellular fluid volumes (calculated from sodium) of the uteri 0 6 and 30 hours after the single dose of estrogen. At 6 hours an increment in intracellular fluid must have been responsible for at least 20 per cent of the total fluid increase. By 30 hours the intracellular fluid has doubled its original volume.

8 8 Estrogen Influence on Electrolytes These findings are consistent with the apparent swelling of cells observed histologically (1). The extent to which the changes observed in the uterus following estrogen stimulation are characteristic of other types of tissue growth remains to be determined. In the case of the uterus the extracellular phase swells 50 to 75 per cent while the cells increase only one-third as much. During this phase the cells take on their normal quota of potassium but definitely less than their quota of phosphorus and probably of solids. This is followed by a phase characterized by marked cell growth during 0 & RS AFTER INJECTION FIG. 3. Changes in average intracellular and extracellular fluid per uterus following administration of 0.1 microgram of cr-estradiol as calculated from chloride and sodium concentrations. The extracellular fluid as estimated from sodium is represented by the area A which lies between Lines 1 and 2 and as estimated from chloride by the area A + B which lies between Lines 1 and 3. The intracellular fluid as calculated from sodium is represented by the area B + C between Lines 2 and 4 and as calculated from chloride is represented by the area C which lies between Lines 3 and which the cells take up approximately constituents. normal amounts of cell SUMMARY An investigation of the electrolyte composition of the immature rat uterus before and after it is stimulated to grow by a single dose of estrogen reveals the following facts. During the first 6 hours after estrogen administration the uterus gains water and chiefly extracellular electrolytes. During this phase there is an increase in the potassium to phosphorus ratio. During

9 Talbot Lowry and Astwood the following 24 hours there is a rapid growth of new protoplasm with a gradual return of the electrolyte pattern to a normal configuration. We wish to thank Professor A. B. Hastings for his valuable suggestions in the preparation of this paper. BIBLIOGRAPHY 1. Astwood E. B. Endocrinology (1938). 2. Hastings A. B. and Eichelberger L. J. Biol. Chem (1937). 3. Fiske C. H. and Subbarow Y. J. Biol. Chem (1925). 4. Manery J. F. and Hastings A. B. J. Biol. Chem (1939).

10 INFLUENCE OF ESTROGEN ON THE ELECTROLYTE PATTERN OF THE IMMATURE RAT UTERUS N. B. Talbot Oliver H. Lowry and E. B. Astwood J. Biol. Chem :1-9. Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references 0 of which can be accessed free at #ref-list-1

THE EFFECT OF ANTICOAGULANTS ON DETERMINA- TIONS OF INORGANIC PHOSPHATE AND PROTEIN IN PLASMA BY OLIVER HENRY GAEBLER

THE EFFECT OF ANTICOAGULANTS ON DETERMINA TIONS OF INORGANIC PHOSPHATE AND PROTEIN IN PLASMA BY OLIVER HENRY GAEBLER (From the Department of Laboratories, Henry Ford Hospital, Detroit) (Received for publication,