Phosphoserine phosphatase: Development formation and hormonal regulation in rat tissues

SRCHIVES

OF

BIOCHEMISTRY

AND

Phosphoserine

BIOPHYSICS

Phosphatase:

Hormonal

of Biological New England

Chemistry, Deaconess

Received

Development

Regulation

S. C. JAMDAR From the Department

134, 228-232 (1969)

AND

Formation

and

in Rat Tissues’

OLGA GREENGARD

Harvard Hospital,

Medical School, and the Cancer Research Institute, Boston, Massachusetts 08216

June 13, 1969; accepted

July 22, 1969

Phosphoserine phosphatase was measured in rat liver and kidney as a function of age, sex, and treatment with different endocrine or metabolic factors. Several differences were found between liver and kidney in the regulation of this enzyme. The phosphoserine phosphatase activity of adult liver was 595 units per g for males, 383 for females, and for adult kidney of both sexes it was 1266. Estrogen treatment raised the level of phosphoserine phosphatase in kidneys after the age of 25 days but not in livers. The high levels of phosphoserine phosphatase in fetal liver, 2500 units per g, decreased to 900 during the last 4 days of gestation and the first 3 postnatal days. During the same time period kidney phosphoserine phosphatase increased from insignificant levels to about 600 units per g. The administration of hydrocortisone (but not of thyroxine, glucagon, or serine) to fetal rats enhanced the decline of liver phosphoserine phosphatase, suggesting that adrenocortical secretion is the stimulus for the normal decrease in liver phosphoserine phosphatase during late fetal life. The injection of hydrocortisone to the pregnant rat also resulted in lower phosphoserine phosphatase in the livers of her fetuses or newborns.

Studies on the biosynthesis of serine from carbohydrate in animals (l-4) and in bacteria (5, 6) had revealed the presence of two possible routes for its formation, the socalled phosphorylated pathway and the nonphosphorylated pathway. Phosphoserine phosphatase (EC 3.1.3.3) catalyzes the last step of the former pathway (7, 8). Recently, Knox et al. (9) investigated the properties and the distribution of this enzyme in different tissues of the rat and in some primary and transplanted tumors. This study indicated the possible importance of the phosphorylated route for serine formation in several normal tissues. Particularly high levels were found in fetal liver, and in a mammary variety of tumors, including 1 This investigation was supported by United States Public Health Service Grant CA 08676-04 from the National Institutes of Health and by United States Atomic Energy Commission contract AT(30-1).3779 with the New England Deaconess Hospital.

carcinomas, even though the normal mammary gland is essentially devoid of phosphoserine phosphatase activity. Previous attempts to alter experimentally the level of this enzyme were restricted to adult rat liver, cultured human cells, and E. coli. In these, serine inhibited both the activity and the synthesis of the enzyme (10) ; the administration of serine or phosphoserine to rats did not alter the level of phosphoserine phosphatase in liver, but a low protein diet caused a small rise (11, 12). The present study compares the developmental formation of phosphoserine phosphatase in liver and kidney and describes its regulation by endocrine factors. MATERIALS

AND

METHODS

Phospho-n-serine was purchased from Sigma Chemicals. The hormones used in this investigation were: hydrocortisone acetate (Merck, Sharp and Dohme) ; Progesterone (Lipo-Lutin, Parke Davis and Co.); and estradiol (Progynon, Scher228

DEVELOPMENT

OF PHOSPHOSERINE

ing). The different age groups of rats were from the NEDH inbred colony. The age of the fetuses was read off a curve of body weight against day of gestation established by Gonzalez (13) which closely agrees with that for our rats. As described previously (14), fetuses in laparotomized mothers were injected intraperitoneally through the uterine wall with the indicated substances. The fetuses were left in situ for 24 or 48 hr. Tissue homogenates (20% in cold 0.15 M KCl) were centrifuged at 150,OOOgfor 30 min. The supernatant fractions were dialyzed at 5” for 2% hr against 0.02 M sodium acetate buffer of pH 6.2, with successive changes of buffer after every 45 min to remove 80% of the inorganic phosphate. The dialyzed preparations were assayed by the method of Knox et al. (9). This method gives a valid measurement of phosphoserine phosphatase concentration; the reaction is specific and proportional to the amount of tissue preparation added and to the time of incubation (9). Each assay consisted of two reaction mixtures (1 ml total volume) in duplicate, one with and one without 0.1 M L-serine, containing also 0.4 ml sodium acetate buffer of pH 6.2, 0.1 ml of 0.1 M MgCl*, 0.1 ml of 0.05 M phospho-D-serine. The mixture was warmed to 37” and the reaction was started by the addition of 0.05 or 0.1 ml of the enzyme preparation. After 60 min 2 ml of 10% trichloroacetic acid was added. Inorganic phosphate in the protein free filtrate was determined by the method of Chen (15) as modified by Ames (16). The difference in phosphate content between the duplicate reaction mixtures with and without L-serine represents the specific phosphoserine

229

PHOSPHATASE TABLE

I

DECREASE OF LIVER PHOSPHOSERINE PHOSPHATASE ACTIVITY UPON PRENATAL ADMINISTRATION OF HYDROCORTISONE A single injection of hydrocortisone (HC) 24-48 hr before assay, was given intraperitoneally to pregnant rats (5 mg/lOO g body weight) or to individual fetuses (0.125 mg/fetus); in the latter case, the injection of hormone was alternated along the uterine horns with those of the vehicle, 0.1 ml saline. Each fetal value is an average of results with two pools (2-6 livers each) from the same litter; the means (&SD) of these average fetal enzyme activities for columns 1, 3, and 4 are 2073 f 246, 1560 + 206, and 1334 f 200, respectively. The postnatal values are means f SD of results of individual livers of neonates of untreated or HC-injected dams. Phosuhoserine hosohatase activity (units/g feta P or neonatal liver) Injections

Age (day of gestation)

To pregnant rats

To fetuses

HC

Wine .I-

19 19.1 19.2 19.3 20.8 21.6 Hours after birth 12

-

2206 2308 2232 1870 1750

1325 f

1176 -

36

(6)

667 f 87 (5)

1861 1442 1487 1711 1280 1579

-

I-

HC

1734 1318 1248 1208 1210 1290

-

phosphatase activity. The enzyme concentrations are expressed in units (nanomoles of inorganic phosphate released per min at 37”) per g of fresh tissue; the value for the standard tissue, adult male rat liver is 595 units f 42 units per g. RESULTS

FIG. 1. Phosphoserine phosphatase activity of rat liver, lung, and brain as a function of age and sex. Each prenatal point refers to a pool of 5-12 retal livers. The postnatal values are means (bracket = 1 SD) of results with 4-8 individual livers (O), brains (O), and lungs (X) of both sexes or of male (0) or female (0) livers.

Fetal rat liver, 3 days before birth, contains over 2200 units/g of phosphoserine phosphatase; i.e., four times more than does adult liver (Fig. 1). It then decreases steadily during the late fetal stage and the first 3 postnatal days to about 900 units. Subsequently, the decrease becomes extremely slow, particularly in males, so that at the age of 70 days a small but significant (p < 0.01) sex difference can be seen, Figure,

23Q

JAMDAR

AND

1 also shows that fetal brain and lung contain almost no phosphoserine phosphatase; in adult rats these organs exhibit significant phosphoserine phosphatase activity but less than half of that seen in liver. Attempts were made to delay or accelerate the decrease of phosphoserine phosphatase level in livers of fetal rats. The administration of serine, thyroxine, or glucagon to fetuses (or of serine to the pregnant rat, 200 mg 3 times daily for 3 days) was without effect but hydrocortisone enhanced the diminution of phosphoserine phosphatase activity (Table I). In these experiments two types of comparisons were made: fetuses from untreated litters were compared with hydrocortisone-injected fetuses and the latter were also compared with their salineinjected littermates. The former comparison (first and last columns in Table I, see also legend) shows clearly that hydrocortisone significantly (p < 0.01) decreased the level of phosphoserine phosphatase in fetal liver. The latter comparison (third and fourth columns) shows only an insignificant effect (p < 0.1). The reason for this is clear from the signifidantly low-er activity (p < 0.01) in saline-injected fetuses (adjacent to those having received hydrocortisone) than in members of uninjected litters (cf. first and third columns). This decrease is not due to the trauma of injection pey se since in litters in which none of the fetuses received hydrocortisone, but were given glucagon, thyroxine, or saline, the levels were identical to those in untreated litters. Thus, the low TABLE THE

LOCK OF EFFECT OF GLUCOSE, E.~RLY POSTNATAL DECREASE

GREENGARD

values in the third column of Table I must mean that some hydrocortisone can reach the “control” fetuses in the same uterus, a phenomenon previously observed in rats (17). It is known that in rats maternal glucocorticoids are transmitted to the fetus; Table I indicates that this is also the case for hydrocortisone injected into pregnant rats. Such rats have fetuses with decreased levels of liver phosphoserine phosphatase, or give birth to rats whose livers contain half as much of the enzyme as do normal newborns. In adult rats, hydrocortisone (2.5 mg/lOO g body weight) did not change the liver phosphoserine phosphatase activity. Attempts were also made (Table II) to interfere with the decrease in the level of liver phosphoserine phosphatase that occurs during the first 3 postnatal days (See Fig. 1). The possibility exists that this phase of diminution is different from that occurring prenatally and is due to the fact of birth, associated with the cessation of the maternal or placental supply of factors. The rapid postnatal rise of several liver enzymes is a consequence of the neonatal hypoglycemia and is inhibited by the administration of glucose (14) ; estrogen was also found to interfere with the developmental formation of at least one enzyme (18). Neonatal rats were now treated with estradiol or progesterone and killed at the age of 72 hr. These agents did not significantly influence the liver phosphoserine phosphatase levels (Table II). Glucose was also without effect. Thus, II

SERINE, ESTRbDIOL, AND PROGESTERONE OF LIVER PHOSPHOSERINE PHOSPHATASE

ON THE

Hormones, dissolved in 0.1 ml of sesame oil, were injected subcutaneously. Glucose and serine, dissolved in 0.1 ml of 0.9% NaCl, were injected intraperitoneally. Control rats received the appropriate vehicle. The levels of phosphoserine phosphatase at the beginning of treatment (24 or O-6 hr after birth) may be seen in Fig. 1. Each value is a mean f SD of results with 5-6 rats. Age of rat (hr) At treatment

24 24 3 2 3

When

assayed

72 72 48 24 7 days

Injection (per rat)

Estradiol, 50 rg Progesterone, 250 pg Glucose, 20 mg 3 times daily L-Serine, 5 mg twice L-Serine, 5 mg twice daily

Phosphwe;sn/[hosThatase Treated

Control

936 981 1417 1479 778

f f f f z!z

154 79 74 27 17

876 810 1335 1385 816

f f + f f

141 114 89 242 16

DEVELOPMENT

OF PHOSPHOSERINE

FIG. 2. Phosphoserine phosphatase activity of rat kidney as a function of age and estrogen treatment. Each prenatal point refers to a pool of 4-12 pairs of kidneys (0). The postnatal points are means (bracket = 1 SD) of results with 3-8 individual rats. When indicated by arrows estradiol (100 pg) was administered once daily for 4 days and assays were done 24 hr after the last injection (4).

the decrease in liver phosphoserine phosphatase, though it overlaps the neonatal state of hypoglycemia, is apparently not caused by it. Increased availability of serine is also an unlikely factor since even prolonged treatment with serine did not enhance the decline (Table II). Figure 2 shows the developmental pattern of phosphoserine phosphatase in kidney. The enzyme level is initially low but it starts to accumulate rapidly at the late fetal stage and continues to rise slowly during the first 40 postnatal days. In kidney, phosphoserine phosphatase activities of adult male and female rats are increased by the administration of estradiol. This increase was smaller in 30day-old rats and did not occur in newborn animals. The same animals that had elevated kidney phosphoserine phosphatase activity upon estradiol treatment (Fig. 2) did not have increased activity in the liver; the values for liver at the age of 5, 30, and 75 days were 863 & S6,674 f 70, and 386 =t 31, respectively, indistinguishable from the control values of 914 f 40,659 f 13, and 390 f 53. DISCUSSION

Significant differences have previously been noted between the rates of develop-

PHOSPHATASE

231

mental formation of the same enzyme in liver and in kidney (19, 20). Phosphoserine phosphatase represents an extreme example of such a difference: during fetal and neonatal life it decreases in the liver from very high to the low adult levels, whereas in the kidney it increases during the same time from insignificant almost to the high adult levels. Too little is known at present about the physiological function of phosphoserine phosphatase and the metabolic states of fetal liver versus kidney to interpret this difference in the enzyme level between the two organs. Additional differences between phosphoserine phosphatase of liver and kidney are the sex difference, seen in liver only, and the positive response to estrogen treatment, exhibited by the kidney enzyme only. The latter phenomenon has also been observed with ornithine aminotransferase: estrogen induces it in kidney (in developing liver it has the opposite effect) and, as in the case of phosphoserine phosphatase, this response to extrogen is not seen in very young animals (18, 19). More than two dozen enzymes have been found first to appear in mammalian liver during late fetal life or at birth and to accumulate to adult levels within a few days (20). Perhaps, an equally large number of enzymes decrease in amount during this period; so far only a few of these have been identified. The mechanisms underlying the process of new enzyme formation have been approached by identifying hormones whose early administration evokes the accumulation of a specific enzyme or a group of enenzymes prematurely (21). It was now attempted to use a similar approach for identifying factors responsible for the decrease in the level of phosphoserine phosphatase during development. An injection of hydrocortisone to fetuses greatly enhanced the decrease of their liver phosphoserine phosphatase levels, suggesting that the secretion of glucocorticoids, known to begin around Day 18 of gestation (22, 23) is the physiological stimulus for the decrease in the level of phosphoserine phosphatase. During the same period of gestation glucocorticoids probably exert the opposite effect on enzymes involved in glycogen synthesis : hydrocortisone causes

232

JAMDAR

AND

premature glycogen deposition in fetal livers (20). Thyroxine that enhances the formation of arginase, NADPH dehydrogenase, and glucose-6-phosphatase (24, 25) and glucagon that evokes tyrosine aminotransferase and serine dehydratase in fetal liver (14), were without effect on phosphoserine phosphatase.’ The mode of action of these hormones on the enzyme-forming system is unknown. The mechanism by which hydrocortisone enhances the developmental decrease of phosphoserine phosphatase is also unknown. It may act indirectly but apparently not through changing the concentration of serine since serine itseIf was without effect. The secretion of corticosterone in the rat declines after the third postnatal day but is high at the time of birth (26) so that the early postnatal decrease in the level of phosphoserine phosphatase, like the prenatal one, is probably also due to glucocorticoids. This decrease is not dependent on the neonatal state of hypoglycemia or on the postpartum diminution of progesterone or estrogen levels since the administration of glucose or of these hormones was without effect on phosphoserine phosphatase. Studies of several enzymes demonstrate that factors important in regulating the levels of an enzyme during development are not identical with those to which the same enzyme responds in the adult tissue. Two of the present observations exemplify this phenomenon: hydrocortisone decreased the level of phosphoserine phosphatase in fetal liver but was without effect on adult liver, and estrogen that raised the enzyme level in the adult kidney did not affect it in young kidney. ACKNOWLEDGMENT We are grateful to Miss Jeanne her skillful technical assistance.

Winsten

for

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