BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

48,

263-274 (1992)

Pyrimidine Nucleotide Synthesis in the Rat Mammary Gland: Changes in the Lactation Cycle and Effects of Diabetes SIRILAKSANA KUNJARA, MILENA SOCHOR, MICHAEL BENNETT, A. LESLIE GREENBAUM, AND PATRICIA MCLEAN Department

of Biochemistry, Building,

University College and Middlesex School Cleveland Street, London WIP 6DB, Great

of Medicine, Britain

Windeyer

Received June 8, 1992 Measurements have been made of the activities of the enzymes of the de nova and salvage pathways of pyrimidine synthesis (carbamoyl phosphate synthetase II (glutamine) (EC 6.3.5.5); dihydroorotate dehydrogenase (EC 1.3.99.11); the overall activity of Complex II (orotate phosphoribosyl pyrophosphate transferase (EC 2.4.2.10) and orotidine Sphosphate decarboxylase (EC 4.1.1.23); uracil phosphoribosyltransferase (EC 2.4.2.9)) in the mammary gland of rats at different stages of the lactation cycle and the effects of diabetes on the activity of these enzymes in lactation have been studied. From a consideration of the changes in enzyme activities and the changes in the tissue concentration of phosphoribosyl pyrophosphate, an activator of the de nova pathway and substrate for both the de nova and salvage routes, it is concluded that the de nova pathway is the major route of pyrimidine synthesis in mammary tissue. Diabetes decreases the activity of the enzymes of the de nova pathway; the effects are particularly marked for Complex II. The present results on pyrimidine synthesis are compared to the pattern for purine synthesis previously published. o IWZ Academic

Press, Inc.

The pattern of metabolic activity in the rat mammary gland over the lactation cycle reflects the changing balance of a variety of processes in the tissue as the initial requirement for precursors of growth slackens in the third week of pregnancy and is replaced by the requirement for precursors of milk synthesis once lactation is initiated. Growth and galactopoiesis involve large changes in the direction and scale of the protein biosynthetic activity and have been shown to be linked to increased nucleic acid metabolism with the tissue DNA increasing some lo-fold from the virgin state to the height of lactation and the RNA content increasing approximately 50-fold over the same time span (1). Increases in the tissue content of soluble ribonucleotides also occur, although on a lesser scale, over this time (2). Changes of this order of magnitude in the nucleotide and nucleic acid levels in the mammary gland can be expected to require concomittant changes in the provision of nucleotide bases for such biosynthesis, and an enhanced activity of the de nova and salvage pathways for the synthesis of the purine nucleotides has been previously reported (3). In that study, a distinctive pattern of enzyme change 263 08854505192 $5.00 Copyright Q 1992 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

264

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ET AL.

was noted in that the de novo pathway appeared to be related to the tissue growth in the period of pregnancy, remaining substantially unchanged from the midpregnancy level through the latter half of pregnancy and lactation, while the enzymes of the salvage pathway increased only late in pregnancy, by which time the development and growth of the gland is virtually completed and then continued to rise during lactation in parallel with milk production. The present study extends this investigation of purine nucleotide metabolism in the mammary gland by following the changes in activity of the enzymes of pyrimidine nucleotide synthesis over the lactation cycle. Pyrimidines are, like the purine nucleotide, involved in growth via DNA and RNA formation but they also have a special role to play in mammary tissue because of the essential role of uridine in the synthesis of lactose and other uridinesugar complexes, and the vital role of cytidine in the synthesis of complex lipids in a tissue which has a high lipogenic activity and turnover of membrane phospholipids resulting from the mechanism of milk secretion. Uridine can be synthesized via the de novo or salvage pathways, the latter involving the recovery of uracil base via a pyrophosphorylation reaction. It seemed important to establish whether a pattern of involvement of these two routes, similar to that previously shown to occur for purine synthesis (3) is also expressed in the mammary gland through the lactation cycle. The de novo pathway of pyrimidine formation is achieved by two cytosolic multienzyme complexes linked by a membrane-bound enzyme located on the outer mitochondrial surface (see 4). The mitochondrial. enzyme is dihydroorotate dehydrogenase (DHODH)’ (EC 1.3.99.11). The first cytosolic complex, which converts ATP, COZ, glutamine, and aspartate to dihydroorotate, is composed of three enzymes, carbamoyl phosphate synthetase II (glutamine) (CPSII) (EC 6.3.5.5), aspartate transcarbamoylase (EC 2.1.3.2) and dihydroorotase (EC 3.5.2.3), which appear to exist in a fixed proportion to one another in the complex (5). Of these three, CPSII is the rate-limiting enzyme and, consequently, only the activity of this enzyme has been measured in the present study on the assumption that the activity of this enzyme can be taken to reflect the overall activity of the entire complex of enzymes. The second complex, which converts orotate to UMP, is composed of two enzymes: orotate phosphoribosyltransferase (OPRTase) (EC 2.4.2.10) and orotidylate decarboxylase (ODCase) (EC 4.1.1.23) (see 4). The recovery pathway of pyrimidine synthesis consists of a single enzyme, uracil phosphoribosyltransferase (UPRTase) (EC 2.4.2.9), and the activity of this enzyme was measured to represent the activity of this route. In view of the role of insulin in regulating the formation of PRPP and the formation of purine nucleotides in the mammary gland (3,6), as well as its wellknown role in sustaining mammary growth and lactation (see 7,8), the effect of 1 Abbreviations used: CPM, carbamoyl phosphate synthetase II (glutamine) (EC 6.355); DHODH, dihydroorotate dehydrogenase (EC 1.3.99.11); the overall activity of Complex II comprising OPRTase, orotate phosphoribosyl pyrophosphate transferase (EC 2.4.2.10), and ODCase, orotidine 5-phosphate decarboxylase (EC 4.1.1.23); UPRTase, uracil phosphoribosyltransferase (EC 2.4.2.9); PRPP, phosphoribosyl pyrophosphate; PPP, pentose phosphate pathway; STZ, streptozotocin.

PYRIMIDINE

SYNTHESIS

IN MAMMARY

diabetes on the activity of both pathways of pyrimidine gland was examined. MATERIALS

GLAND

265

synthesis in the mammary

AND METHODS

Materials NaH14C03 (50-60 Ci/mol) and [methyZ-3H]thymidine were obtained from Amersham International (Amersham, Bucks, UK). Scintillation fluids, Optiphase, Ecosint 0, and Filtron-X, were obtained from National Diagnostics (Aylesbury, Bucks, UK). The cellulose monoacetate strips (Celagram II) were obtained from Shandon Southern (Runcorn, Cheshire, UK). Animals Albino rats of the Wistar strain, weighing 220-240 g before mating and undergoing their first pregnancy, were used. They were taken at intervals, shown below, in pregnancy and lactation, and at 3 days after removal of pups, i.e., in mammary involution, and were killed by cervical dislocation. The three abdominal glands of one side were removed and used for the preparation of tissue slices for thymidine incorporation or extracts for enzyme assays. In order to study the effects of diabetes, animals were taken on the 7th day of lactation and given an intravenous injection of streptozotocin (60 mg/kg body wt). They were killed 3 days later. Enzyme Determinations 1. Carbamoyl phosphate synthetase II. The tissue was homogenized (1: 3) in an extraction buffer containing 0.02 M Tris, 0.15 M KCl, 0.2 mM EDTA, 0.2 mM dithiothreitol, pH 8.2, and containing 30% dimethyl sulfoxide and 5% glycerol. The clear supernatant obtained after centrifugation at 105,OOOg for 30 min was used immediately without further treatment. The assay procedure was as described by Tatibana and Shigesada (10) and the incubation time was 15 min at 37°C. The reaction was stopped by the addition of 0.2 ml 50% acetic acid and the whole mixture evaporated to dryness at 110°C to remove all acid labile compounds. The resultant cake was resuspended in 0.2 ml of 10% H202 and 0.5 ml water. After the addition of 10 ml of the scintillator Ecosint A, the vial was counted in a Beckmann LS7500 counter. 2. Dihydroorotate dehydrogenase. The tissue was homogenized (1: 10) in 0.25 M sucrose/O.01 M Tris, pH 7.4, and then centrifuged at 600g for 10 min. The supematant from this was centrifuged at 105,OOOg for 30 min and the pellet thus obtained was resuspended in 4 ml of the sucrose/T& buffer to give a 1: 4 tissue suspension. This suspension was then freeze-thawed three times to disrupt the mitochondrial membrane. Finally, the preparation was dialyzed twice against 100 vol of buffer, each time for 30 min. The assay procedure was as described by Peters et al. (11). The reaction time was 10 min and the temperature 37°C. The reaction was stopped with trichloracetic acid as described by Peters et al. (11) and the protein-free supematant from this was used for the measurement of the erotic acid formed by spectrophotometry at 285 nm, taking a value of 6.72 x lo6 as the molar extinction coefficient.

266

KUNJARA

ET AL.

3. Complex ZZ, orotate phosphoribosyl pyrophosphate transferase and orotidine 5’-phosphate decarboxylase. The tissue was homogenized (1: 10) in 0.25 M su-

crose/0.02 M triethanolamine/O.l mM dithiothreitol, pH 7.4, and then centrifuged at 25,000g for 30 min. The supernatant was dialyzed against 100 vol of the original sucrose buffer for 40 min. The assayof the overall activity of OPRT and ODCase (the Complex II) was as described by Jones (12). The reaction was continued for 15 min at 37°C and was carried out in a reaction vessel which included a tube containing 0.5 ml of 1 M hyamine as described by Kunjara et al. (13) to collect the 14C02produced. After 40 min to allow complete absorption, the,hyamine was quantitatively transferred to a scintillation vial containing 4 ml Ecosint 0 scintillation fluid and counted in a Beckmann LS 7500 counter. 4. Uracil phosphoribosyltransferase. The tissue was homogenized (1:5) in a buffer consisting of 0.25 M Tris/O.O2 M KC1/6 mM MgC&/l mM dithiothreitol, pH 8.0, and then centrifuged at 105,OOOgfor 30 min. Dialysis did not affect either the activity or the blank and was, therefore, omitted. The assay was as described by Kizaki and Sakurada (14) except that 5’-fluorouracil was used as substrate and 3 mM thymidine triphosphate was included to inhibit nucleotidases (15). The 5’-fluoro[6-‘4C]uracil content for each assaywas 0.25 PCi of a preparation that contained 56 Ci/mol. The use of 5’-fluorouracil as substrate was based on the report by Reyes (16) that both uracil and the fluoro derivative were pyrophosphorylated by the same pyrimidine pyrophosphorylase and the fluoro derivative produced some 50 times more product than the natural base at the optimal pH of 10. The incubation time was 20 min at 37°C and the reaction was stopped by the addition of 4 N formic acid. Separation of the product from substrate was achieved by the electrophoresis, in 0.1 M Tris buffer, pH 8.0, at 150 V, at 4°C for 90 min, of 5 ~1 of the incubation mixture, overlaid on 5 ~1 of a 1: 1 carrier mix of 2 mM UMP and 2 mM 5’fluorouracil, on cellulose monoacetate strips (2.5 x 12 cm). After 90 min the strips were collected, air-dried, and divided into 11 x l-cm bands which were then counted in scintillation vials containing 4 ml Filtron-X scintillation fluid. Recovery of counts after electrophoresis was 88-93%. The activity of the enzymesis expressed as micromoles per hour, for the total gland, with the mammary gland values in Table 2 corrected for the presence of retained milk (17). Thymidine

Zncorporation

into DNA

Approximately 300 mg of mammary gland slices,cut from the abdominal glands, was incubated in 5 ml of Eagle’s minimal essential medium containing Earle’s balanced salt solution (GIBCO 041-1010) and sufficient KHzP04, pH 7.4, to bring the final phosphate concentration to 10 mu. L-Glutamine was added to give a final concentration in the medium of 2 mM. The glucose concentration was 20 mM. The substrate for incorporation was 5 &i [methyZ-3H]thymidine (49 Ci/mmol). The flasks were gassedwith Oz/COz (95/5) and the total incubation time was 60 min. After incubation the slices were collected and washed twice with 5 ml cold 0.9% NaCl. The washed slices were transferred to an homogenizing vessel con-

PYRIMIDINE

SYNTHESIS

IN MAMMARY

GLAND

267

taming 3 ml of 0.6 N HC104 and were homogenized with an Ultra-Turrax blender. They were then stood on ice for 30 min and the congealed surface fat was removed. The nucleic acids were collected by centrifugation at 2000g for 10 min and washed five times with 0.2 N HC104. After the pellet was resuspended in 3 ml of 1 N HC104, the nucleic acids were hydrolyzed by heating at 90°C for 20 min. This hydrolysate was again centrifuged at 2000g for 10 min and 1 ml of the supernatant was taken for counting in 10 ml of Optiphase scintillator. The RNA, DNA, and protein contents of the tissue were estimated as previously described (3) with purified tRNA and calf thymus DNA (Sigma) as standards. RESULTS The data shown in Table 1 record a progressive increase in mammary gland weight over the entire lactation cycle, although such weights are inflated by the presence of retained milk in lactation and, in particular, after weaning. Examination of the DNA content of the tissue (Table 1) relative to the gland weight (corrected for the presence of retained milk) reveals that there is an increasing cellularity of the gland in pregnancy, especially in the preparturient period and that this higher level is maintained in the first half of lactation. The cellularity is again increased in the second half of lactation, but is sharply reduced by weaning or diabetes. The tissue RNA follows a similar course, although the rise of RNA in the second half of lactation is more rapid than is that of DNA. At the end of lactation the RNA content is almost 35 times the level found at the earliest stage of pregnancy examined and some 3.5 times greater than that found at the beginning of lactation. Incorporation of Labeled Lactation Cycle

Thymidine

into DNA in the Mammary

Gland over the

The incorporation of thymidine into DNA increases some eightfold during pregnancy. There is a further rise in early lactation but incorporation decreases very sharply such that it has returned to the 5day pregnancy value by the end of lactation. Diabetes causes a dramatic fall in the rate of this incorporation (Table 1). Enzymes Pyrimidine

Synthesis in the Mammary

Gland

The changes in the activity of these enzymes over the course of the lactation cycle are shown in Table 2. (i) The de novo pathway. Although a general overall pattern of increasing activity of the enzymes of this pathway appears to exist, significant differences occur between the behaviors of the component enzymes, with respect to the time of appearance and the scale of the increase, which may be of importance in relation to the sequence of events occurring in the gland. (a) CPS II: The activity of this enzyme increases over the first 15 days of pregnancy. The onset of milk production is accompanied by a further increase in activity which then continues to rise over the remainder of lactation. Weaning causes a sharp fall in activity. (b) Dihydroorotate dehydrogenase: The most striking change in the activity of

268

KUNJABA

ET AL.

TABLE 1 Vital Data, Nucleic Acid Content, and Incorporation of Thymidme into Mammary Gland of Bats during the Lactation Cycle and the Effect of Diabetes

Stage Pregnancy 5 days

BUY weight k)

Mammary gland weight” cd

241 + 12

0.73 + 0.05

(6)

10 days 15 days 20 days Lactation 1 day 5 days 10 days 15 days 20 days Weaning 3 days Diabetes 7L + 3Dt (% of lo-day value)

(6)

Mammary Mammary gland Incorporation of gland RNA DNA thymidine (mg/total gland)b(mg/totaI gland)b(pmol/total gIand/h)b 2.84 f 0.06

1.29 k 0.25

(6)

(6)

262 + 5 (5) 270 f 12 (5) 331 f 15 (5)

2.12 * 0.16 (5) 2.67 f 0.42 (5) 4.18 f 0.36 (5)

6.36 f 0.66 (5) 18.80 + 2.30 (5) 24.60 f 3.12

262 2 11

2.79 f 0.31

27.37 f 1.36

(8)

271 f 14

(8)

299 f 15

(6)

295 + 17

(6)

306 f 10

(6) 266 + 9

(6) 224 k 14 (5) 75%**

(8)

3.49 f 0.29

(8)

4.14 + 0.26

(6)

4.31 + 0.28

(6)

4.60 f 0.33

(6) 5.50 + 0.42

(6) 2.41 f 0.27 (5) 58%**

(6)

3.52 2 0.61 (5) 4.35 + 0.88 (5) 6.53 + 1.21

(6)

11.5 (5) 46.4 (4) 81.4 (4) 94.9 (4)

+ 1.8 + 4.4 k 18.9 + 7.9

6.33 2 0.53

153 f 28

(8)

(8)

(8)

03)

(8)

34.41 f 3.63 68.60 k 7.58

(6)

86.96 f 10.7

(6)

97.75 f 6.26

(6) 39.22 2 4.13

(6) 17.06 -c 0.87 (8) 25%***

6.49 k 0.42 10.23 k 0.91

(6)

10.43 IL 0.78

(6)

10.90 2 0.92

(6) 6.53 zt 0.61

41.8 f 6.5 (4) 33.4 + 0.5 (4) 14.5 + 0.60

(6)

17.0 + 1.8 (4) -

(6) 4.46 ” 0.36 (8) 44%***

3.97 f 1.32 (5) 13%***

Note. The values are given as means f SEM, and the numbers of observations are given in parentheses. Fisher’s P values for the comparison of the 3 days of diabetes, following STZ treatment of 7-day lactating rats (t7L + 3D), with the normal lo-day lactation values are shown by asterisks: **p < 0.01; ***p < 0.001. ’ Total weight of the three abdominal glands of one side, uncorrected for the presence of retained milk. b Content of RNA or DNA, or thymidine incorporation, the three abdominal glands of one side.

this mitochondrial-linked enzyme occurs in lactation where the rise is sharper, and greater, than that found for CPS II or for Complex II, observations which suggest that the activity of the enzyme might be more closely related to the increase in mitochondrial numbers and oxidative capacity which occurs in mammary gland as pregnancy and lactation advance. There is an excellent correlation between the values for DHODH activity given here and the values for succinoxidase activity reported by Greenbaum and Slater (18). (c) Complex II: This enzyme follows a pattern of change similar to that of CPS

PYRIMIDINE

SYNTHESIS

IN MAMMARY

269

GLAND

TABLE 2 The Activity of Enzymes of the Pathways of Pyrimidine Synthesis in Mammary Gland at Different Stages of the Lactation Cycle and the Effect of Diabetes

Stage

Carbamoyl phosphate synthetase II

Dihydroorotate dehydrogenase pmol/total

Pregnancy 5 days 10 days 15 days 20 days Lactation 1 day 5 days 10 days 15 days 20 days Weaning 3 days (% of 20 days) Diabetes 7L + 3Dt (% of 10 days)

Complex II

Uridine phosphoribosyltransferase

gland/h

0.061 0.444 0.607 0.604

t f 2 -t

0.007 0.009 0.066 0.025

0.840 0.694 1.943 3.00

+ + + *

0.113 0.119 0.232 0.54

0.126 0.123 0.483 0.422

2 f -t f

0.025 0.006 0.085 0.046

12.27 16.26 21.60 18.57

f f f 2

1.68 2.00 0.73 1.47

0.791 0.796 1.155 1.116 1.141

2 k + + +

0.113 0.067 0.153 0.194 0.102

6.20 14.68 30.22 29.67 36.72

+ ” ” + +

1.02 1.20 4.18 3.36 4.31

0.397 0.695 1.460 0.572 0.446

+ + 2 f f

0.052 0.113 0.151 0.064 0.043

20.20 19.92 34.51 26.00 28.79

f f k f 2

3.10 1.54 2.71 2.43 3.53

0.169 ? 0.025 15%***

9.69 k 1.25 26%***

0.680 k 0.090 152%*

66.65 f 6.04 230%*‘*

0.699 ZL 0.059 61%**

7.16 f 0.81 24%‘**

0.093 2 0.028 6%***

15.13 2 2.94 44%***

Note. The values are given as means + SEM; the numbers of observations are the same as those given in Table 1. Fisher’s P values for the comparison of enzymatic changes in weaning with the activity in 20-day lactating rat mammary gland and for the comparison of 3 days of diabetes, following STZ treatment of 7-day lactating rats (t7L + 3D), with the normal IO-day lactation values are shown by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001. ’ Activity in the three abdominal glands of one side.

II except that the maximum activity is achieved at midlactation and then diminishes rapidly. It is also significant that removal of the pups does not lead to a further decrease in activity such as occurred with CPS II and DHODH. It should be noted that, in the mammary gland, the overall activity of Complex II is close to that of CPS II, the rate-limiting enzyme, in contrast to the more general pattern of tissues where the activity of Complex II exceeds that of CPS II (4,12). (ii) The salvage route. The activity of UPRTase, the sole enzyme of this pathway, is substantially higher than that of either of the enzymes of the de nova route but rises only slowly in pregnancy and lactation, much less than the enzymes of the de lzovo route. Again, in contrast to the enzymes of the de ltovo pathway there is a highly significant increase in the activity of UPRTase following removal of the pups. The Effect of Diabetes on the Activity the Mammary Gland

of the Enzymes of Pyrimidine Synthesis in

This was studied in animals which had been injected with STZ on the 7th day of lactation and killed 3 days later. In Table 2 the enzyme activities in the glands

270

KUNJARA

ET AL.

of such treated rats are compared to those in the untreated lo-day lactating animals. In animals made diabetic in this way, lactation, as judged by the growth curve of litter weight, has virtually ceased (6). In the present study, diabetes dramatically decreased the activity of each of the enzymes studied, although the extent of the fall varied, falling by 40% in the case of CPS II, 76% for DHODH, and 94% in the case of Complex II. The activity of UPRTase falls to approximately half of its lo-day lactation value (Table 2). Of particular import is the extreme sensitivity to diabetes of the enzymes of Complex II, of which less than 10% of the activity is measurable in the lactating rat mammary gland 3 days after STZ treatment (Table 2). In seeking an explanation for this major loss of activity, measurements were made of the individual activities of OPRTase and ODCase, the two component enzymes of the complex. It was found that 3 days after STZ treatment OPRTase fell to 44% and ODCase to 25% of the values found at 10 days of lactation (results not shown). It is known that, in a number of tissues, metabolites of the enzyme complex are channeled and that the steady-state concentration of OMP, reported as 0.05-0.10 PM, is substantially lower than the K, for ODCase of 0.3 PM (12), although it should be noted that the low tissue value for OMP refers to the cellular concentration and not to the local concentration at the interface between OPRTase and ODCase, which could be substantially higher. Thus, one hypothesis that can be advanced to explain the more marked inhibition of Complex II relative to the activities of the individual component parts is that the decreased rate of formation of OMP via OPRTase has a significant ongoing effect on the rate of ODCase, and this substrate limitation could reinforce the effect of the decline in ODCase activity itself. Other possibilities exist, for example, that the state of aggregation of the enzyme could vary through mono-, di-, and tetrameric forms as has been reported to occur in other cells, and these forms have K, values for OMP ranging over a lOO-fold difference (20). Thus, changes in the intracellular milieu, or as a result of susceptibility to extraction procedures, could exert profound effects on the measured enzyme activity. The effect of diabetes on the properties of Complex II could be important to an appreciation of the role of this second enzyme complex in the control of lactation. DISCUSSION Pyrimidine

Nucleotide

Synthesis in the Lactation

Cycle

The changing pattern related to growth and milk formation in the mammary gland can be expected to be reflected in variations in the nucleotide metabolism of the tissue as demands for RNA, DNA, ATP, and UTP alter in the different phases of the lactation cycle. The IO-fold rise of RNA in pregnancy, and the further 4-fold rise in lactation (Table l), and the increase in the tissue content of adenine and uridine nucleotides, as well as of the uridine-sugar derivatives, from late pregnancy to late lactation (by 3- and 8-fold, respectively) (2), all indicate the magnitude of the alteration of biosynthetic activity. In addition, there is a substantial loss of uridine during lactation consequent to the process of milk secretion (2) and this must also be replaced.

PYRIMIDINE

SYNTHESIS

IN MAMMARY

GLAND

271

Among the many factors involved in the regulation of the pyrimidine pathways, two are addressed here: First, the changes in the activity of the component enzymes of the de nova and salvage pathways of pyrimidine synthesis and, second, the modifying effects of PRPP as an activator and substrate on the activity of these enzymes. In the de lzovo pathway, CPS II increases dramatically in pregnancy (by some lOO-fold), but only 2-fold in lactation and Complex II follows a similar, but less marked, pattern of change. The salvage pathway enzyme, UPRTase, stands in sharp contrast in that there is relatively little change in activity over the whole lactation cycle. The contribution of these enzyme changes to the overall nucleotide synthetic activity of the gland is strongly modified by concurrent changes in PRPP concentration and the respective K,,, and K, values of the enzymes for which this is a substrate and activator. In the formation of PRPP, the activity of PRPP synthetase increases lo-fold over pregnancy and by an additional factor of 3-fold in lactation; the tissue content of PRPP increases from 0.028 nmol/g at 5 days of pregnancy to 10.2 nmol/g at midlactation (6). Since the K, of CPS II for PRPP is 4-9 PM (19), the effect of such changes would be to accentuate the increase in enzyme activity strongly, particularly from late pregnancy onward, as the PRPP content reaches the critical level for CPS II activation. The PRPP changes would have the effect of progressively raising the activity of CPS II, bringing it more in line with the changes in the rate of milk formation in lactation. Similarly, Complex II activity would also be sharply modified by PRPP since the K,,, of OPRTase is 15-16 PM (12,20). The enhancing effect of increased PRPP content on the activity of these two enzymes serves to bring them more into line with the measured activity of DHODH, which increases progressively throughout the lactation cycle (Table 2). Although the measured activity of UPRTase is high and appears to indicate that the salvage pathway has a substantial role to play in the synthesis of pyrimidines in the mammary gland, the significance of this route of pyrimidine synthesis in the mammary gland is open to question. The activity of UPRTase, when measured with its natural substrate, uracil, is some 50 times less than that found with 5’-fluorouracil (16) used in the assay and, on this basis, the values recorded in Table 2 are considerably higher than those that could be expected in vivo. In addition, the K, of UPRTase for PRPP is 120 PM (16), a value far in excess of the tissue content of this metabolite. These two factors would set the in vivo activity of this enzyme to very low levels and thus diminish its apparent importance. Three other facts also indicate a low significance for this pathway: (i) the variations in the activity of the enzyme in the lactation cycle are relatively minor compared to the changes in the activity of the enzymes of the de l~ovo route, and there is little correlation between the salvage route and the developing biosynthetic activity of the gland (Table 2); (ii) there is a rise in the activity of UPRTase in weaning, i.e., at a time when the biosynthetic activity of the gland has virtually ceased. It is probable that this latter manifestation is related to the tissue remodeling that occurs at this stage of the lactation cycle and, in particular, after weaning (21); (ii) the finding that diabetes, which has a profound effect on lactation (see below),

272 has a lesser effect on UPRTase pathway (Table 2).

KUNJARA

ET AL.

activity

than on the enzymes of the de nova

Comparison of Enzyme Projiles of Pyrimidine and Puke Synthesis The comparison of the pattern of change of the enzymes of purine synthesis previously reported (3) with those of pyrimidine synthesis reported here reveals a striking parallelism in response to the first steps of the de novo routes of each pathway. In purine synthesis, the most marked increase in phosphoribosyl pyrophosphate amidotransferase activity, measured under optimal conditions in vitro, is in the period of mammary growth in pregnancy (50-fold); in lactation, the activity of this first, and rate-limiting, step is essentially constant (3). The effect of increasing the substrate, PRPP, may again be an important factor in modifying the activity of this enzyme, although the very high & of 250 PM (22) would probably serve to maintain its activity at a relatively low level throughout the lactation cycle despite the rising concentration of PRPP. On the other hand, the enzymes of the salvage route, adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) show a high level of activity relative to the de novo route and their lower K,,, values (in the region of 5-6 PM (23,24), and the progressive increases in the activity of the enzymes during lactation, all point to a central role for the salvage route in the synthesis of purines during the lactation cycle. Thus, despite the resemblances between the developmental patterns of the enzymes of the de novo pathway of purine and pyrimidine metabolism, their contribution to nucleotide synthesis in the mammary gland may be very different. On the basis of the enzyme profiles discussed above, the pattern of bioavailability of PRPP at various stages of the lactation cycle and the kinetic constants of the component enzymes of nucleotide synthesis for PRPP, the results may be interpreted to indicate that, while the salvage route may be the more important pathway for purine synthesis, the de novo pathway predominates in pyrimidine synthesis. EfSect of Diabetes on Pathways of Pyrimidine

Synthesis in Lactation

A spectrum of hormones regulate the growth and metabolism of the mammary gland at different stages of the lactation cycle, the most significant in lactation being insulin, glucocorticoids, and prolactin (25). The extreme sensitivity to insulin is shown by the eightfold stimulation by insulin of glucose uptake by the mammary gland, an effect which is reported to be higher than for any other tissue thus far studied (26,27). Similarly, the sharp decline in lactational performance, of the tissue RNA content and of the activity of enzymes forming ribose 5-phosphate and PRPP in lactating mammary gland after induction of diabetes, has also been established. There is also a fall in the activity of enzymes of the salvage pathway of purine synthesis (APRT by 46% and HPRT by 76%) (3,6), while the key enzyme of the de novo pathway of purine synthesis remains unchanged (3). The present results on the effect of diabetes, induced during lactation, on pyrimidine synthesis, complement the findings in relation to purine synthesis by showing highly significant decreases in the activity of the enzymes involved. However, in sharp contrast to the observations on the purine pathways, it is in the de nova route that the major changes occur, the salvage route remaining relatively unchanged (Table 2).

PYRIMIDINE

SYNTHESIS

IN MAMMARY

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273

The remarkable decrease in the activity of Complex II in diabetes (to 6% of its value at the corresponding period of lactation) may be of particular significance. Diabetes of 3 days duration, initiated on the 7th day of lactation, diminishes milk secretion virtually to zero, as judged by changes in litter weight (6), but has far less effect on the activity of CPS II. However, the effect of diabetes on Complex II is so sharp that this second complex of the de nova pathway is reduced to about 15% of the activity of the first, and generally regarded as the rate-limiting, complex of pyrimidine synthesis and, on this basis, may well be the principal factor for the cessation of lactation. In considering the effect of STZ diabetes on the metabolism of lactating mammary gland, the sevenfold decrease in prolactin that has been shown to occur as early as 2 h after administration of STZ (28) introduces a potential complication. Further studies on the effect of prolactin on pathways of pyrimidine synthesis seem merited. The cascade of events following the induction of lactation and their “switch off’ in diabetes shows a coordinated pattern in which hormonal effects are paramount, insulin and IGF-1 playing an important role in pregnancy and insulin in lactation (25,26,29). Included among the wider changes associated with these hormonal shifts are alterations in glucose uptake, metabolism of glucose by the pentose phosphate pathway (the latter yielding ribose moieties for PRPP formation), alterations in enzymes of the salvage routes of purine synthesis and de ~OVO route of pyrimidine synthesis, each, in turn, linked to RNA synthesis and the formation of milk products. It is proposed that this pattern of events is highly coordinated and that the regulation of PRPP synthesis is central to the integrated control of nucleotide synthesis and, thus, of major biosynthetic routes in lactation. ACKNOWLEDGMENTS We thank the Basil Samuel Charitable Trust, the Association for International and the British Diabetic Association for grants in support of this work.

Cancer Research,

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Pyrimidine nucleotide synthesis in the rat mammary gland: changes in the lactation cycle and effects of diabetes.

Measurements have been made of the activities of the enzymes of the de novo and salvage pathways of pyrimidine synthesis (carbamoyl phosphate syntheta...
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