Comp. Biochem. Physiol. Vol. 103B,No. 1, pp. 113-118, 1992 Printed in Great Britain
0305-0491/92 $5.00 + 0.00 © 1992Pergamon Press Ltd
CHANGES IN APYRASE ACTIVITY IN UTERUS A N D M A M M A R Y G L A N D D U R I N G THE LACTOGENIC CYCLE M. A. VALENZUELA,L. COLLADOS,A. M. KET'rLUN, M. MANCILLA,H. LARA, J. PUENTE, E. ARANDA,*L. CHAYET,A. ALVAREZ and A. TRAVERSO-CORI Departamento de Bioqulmica y Biologia Molecular, Facultad de Ciencias Qulmicas y Farmac~uticas, Universidad de Chile, Casilla 233-Santiago I, Chile (Fax: 562-2227900); and *Departamento de Hematologia, Facultad de Medicina, Pontificia Universidad Cat61ica, Santiago, Chile (Received 6 February 1992; accepted 6 March 1992)
Abstract--1. The purpose of this present research was to explore the possible roles of ATP-diphosphohydrolase (apyrase) in two tissues with high energetic demands during cell proliferation and differentiation. 2. Changes in apyrase activities during the pregnancy lactation cycle were examined in the rat uterus and mammary gland. 3. A significant decrease in apyrase activity (ATPase-ADPase) was observed in the pregnant uterus; this observation correlates with a minor inhibitory effect on platelet aggregation. 4. In mammary gland, the enzyme activity increases during lactation in parallel with an increase in blood supply, synthesis of glycoproteins and cell proliferation. 5. Apyrase activity did not change during the estrous cycle. Estradiol administration to rats slightly increased (20%) both ATPase-ADPase activities. 6. The probable function of apyrase is finally discussed, based on its substrate specifity and subcellular localization.
INTRODUCTION The presence of an ATPase-ADPase activity corresponding to ATP-diphosphohydrolase (EC 3.6.1.5), or apyrase, has been shown in several animal cells and tissues (Liithje et al., 1988; Valenzuela et al., 1989; Schadeck et al., 1989; Papamarcaki and Tsolas, 1990; Pieber et al., 1991). The enzyme is principally located in the microsomal fraction, and its activity depends on bivalent cations, Ca 2÷ being the most effective. This ATPase-ADPase activity is inhibited neither by oligomycin nor by ouabain, specific inhibitors of mitochondrial and (Na ÷, K+)-ATPase, respectively (LeBel et al., 1980; Knowles et al., 1983; Miura et aL, 1987; Valenzuela et al., 1989, Papamarcaki and Tsolas, 1990). The ability of the enzyme to hydrolyze both ATP and A D P to A M P and inorganic phosphate has suggested a functional role for ATP-diphosphohydrolase in platelet aggregation. This suggestion is based mainly on the extracellular localization of the enzyme in the plasma membrane of the aortic epithelium (Miura et al., 1987; Yagi et al., 1989) and as an ectoenzyme in erythrocytes (Liithje et al., 1988). This possibility is also supported by Hutton et al. (1980) who found that fresh myometrial extracts containing ADPase activity (which, at the present, we know must correspond to apyrase) inhibited platelet aggregation in a concentration-dependent manner. Recently, we have also found a similar effect in a microsomal fraction of rat placenta (Pieber et al., 1991). Due to the high metabolic activity and differentiation of the rat uterus and mammary gland during the pregnancy-lactation cycle, many studies have been focused on ATPase activities present in these
tissues. Although several ATPases have been described in these tissues their relationship to apyrase and its physiological role is unknown (Carraway et al., 1980; Enyedi et aL, 1988; Missaen et al., 1988). Some of the enzymes described are responsible for the ATP-dependent Ca2+-uptake, and a Ca2+/Mg 2+stimulated ATPase has also been identified, but no physiological function for this activity has yet been proposed. The enzyme described in this paper (an apyrase) does not correspond to the ATPase involved in Ca 2+ uptake, because they differ significantly in substrate and bivalent metal ion specificity and in insensitivity to sulfhydryl reagents (Enyedi et al., 1988; Valenzuela et aL, 1989). In the present paper we present evidence of the changes occurring to this enzyme with ATPaseADPase activities in rat uterus and mammary gland during pregnancy-lactation and estrous cycles and the putative role of this enzyme in the physiology of these organs based on its subcellular localization and specificity on nucleoside di- and triphosphates.
MATERIAL AND METHODS
Animals Adult Sprague-Dawley rats (weight 250-200 g) produced in our facilities were housed under controlled conditions of photoperiod (12L: 121)) and temperature (22°C) and were fed with rat chow pellets ad libitum. Estradiol treatment was initiated by subcutaneous implantation of a silastic capsule (20ram in length/100g body weight; o.d, 2.16ram; i.d, 1 ram) containing estradioi dissolved in corn oil at 200#g/ml, a concentration that produces normal serum levels at proestrus (Ojeda et al., 1986).
113
114
M.A. VALENZUELAet al.
Enzyme assay Apyrase activity was followed with ATP (ATPase activity) or ADP (ADPase activity) as substrates (final concentration, 2 mM) in the presence of 5 mM CaCI z in 100 mM Tris HCI, pH 8.0. Pi released was determined by the method of Fiske and SubbaRow (1925). A unit of activity (U) is equivalent to 1 #mol of Pi liberated per min at 30°C. The activity of other nucleoside di- and triphosphate was tested under the same conditions. The true ATPase and ADPase activities corresponding to apyrase were measured in homogenates substracting the possible presence of mitochondrial ATPase, (Na ÷, K +) ATPase and adenylate kinase through the effect of their specific inhibitors. The presence of mitochondrial ATPase in pregnant uterus and lactating mammary gland could be demonstrated by the inhibitory effect of oligomycin (0.9 mg/ml), but the activity was low in comparison to that of apyrase which constituted about 80% of ATPase activity measured. Oligomycin did not affect the activity of the homogenates of both virgin tissues. (Na +, K ÷ )-ATPase was not detected, as indicated by the lack of effect of 5 mM ouabain. The adenylate kinase inhibitor, Ap5A (Feldaus et al., 1975) inhibited apparent ADPase activity by 10% throughout, so the possibility that this activity that this activity arise from a combination of ATPase with adenylate kinase can be discarded.
Protein determination Protein concentrations were measured by the method of Lowry et al. (1951) using BSA as standard.
DNA determination DNA levels were determined according to Burton (1956).
Subcellular fractionation The subcellular fractionation procedure was as previously described (Valenzuela et al., 1989).
Further purification of the microsomal fraction Mammary gland, from rats in pregnancy or lactancy, (between 5 and 6 g) were fractionated to obtain microsomal fraction as already mentioned. The 100,000g pellet was suspended in 2.5 ml of the homogenization buffer. This suspension, containing 8.5% sucrose, 25 mM KC1, 50 mM Tris-HC1, pH 7.5, was placed at the top. of 6 ml of each of the following sucrose layers dissolved in 50 mM Tris-HC1, pH 7.5: 60, 50, 38.7 and 29%. The centrifugation was done in a Beckman Spinco Model L centrifuge with a SW 25 rotor for 60 min at 40,100 g. The fractions of the interfaces 8.2/29, 29/38.7 and 50/60 containing the membrane bands were collected and diluted with distilled water to 8.5% sucrose and centrifuged at 100,000g for 60 min. A small pellet was also obtained at the bottom of the gradient and this fraction and the resulting pellets, after the last centrifugation, were suspended in 0.5 ml of the homogenization buffer. The 5'-nucleotidase and glucose-6-phosphatase, measured as described by Beaufay et al. (1974), were used as plasma membrane and endoplasmic reticulum marker, respectively. The Golgi apparatus was detected through the galactosyl transferase according to Fleisher and Kewina (1974).
Platelet aggregation Platelet aggregation was monitored in an aggregometer PAP-4 (Bio-Data Corp.) using citrate-treated platelet-rich plasma according to Born (Born, 1962). A final concentration of 3/~M of ADP was added to induce aggregation. The decrease in light scattering was followed and results expressed as a percentage of the values found in the controls where tissue samples were replaced by either the homogenization buffer or 0.15 M NaC1.
RESULTS
Substrate specificity o f uterine and mammary microsomal fractions M i c r o s o m a l fractions of b o t h tissues with none of the interfering enzymatic activities (Valenzuela et al., 1989) m e n t i o n e d in Materials a n d M e t h o d s , were used to determine substrate specificity. The fractions of b o t h tissues isolated from virgin rats showed a r a t h e r b r o a d specificity towards nucleosides di- a n d t r i p h o s p h a t e s (Table 1). The higher action o n the t r i p h o s p h a t e derivatives is more i m p o r t a n t with the uterine sample.
Further purification o f microsomal./?actions The different types of m e m b r a n e s of the microsomal pellet were partially separated by discontinuous sucrose gradient. The distribution o f apyrase was n o t different w h e n the gland was isolated from the pregnancy period or from the lactation period. The d a t a s h o w n c o r r e s p o n d to 4 days o f lactation. Purity a n d recovery o f the isolated m e m b r a n e fractions were determined by the use of enzymatic markers. The A T P a s e - A D P a s e activities c o r r e s p o n d i n g to apyrase were f o u n d p r e d o m i n a n t l y at the interfaces 8 . 5 - 2 9 % ( F r a c t i o n A) a n d 2 9 - 3 8 . 7 % ( F r a c t i o n B) of the sucrose gradient. Table 2 shows t h a t the e n r i c h m e n t in specific activities a n d distribution of apyrase parallel the 5' nucleotidase activity. N o glucose-6-phosphatase were detected in these m e m b r a n e fractions, discarding the association o f apyrase to the endoplasmic reticulum. A l t h o u g h these fractions are highly c o n t a m i n a t e d with Golgi apparatus, since galactosyl transferase activity was detected, a nucleoside d i p h o s p h a t a s e different from apyrase has been described in this organelle ( N o v i k o f f a n d Goldfischer, 1961; K u h n a n d White, 1977). The c o n t a m i n a t i o n with other ATPases or adenylate kinase in fractions A a n d B m a y be ruled o u t because apyrase activity of these fractions was u n c h a n g e d when olygomicin, o u a b a i n or A p 5 A were added.
Microsomal distribution o f apyrase during the pregnancy-lactogenie cycle A T P a s e a n d A D P a s e activities showed a microsomal distribution b o t h in uterus a n d m a m m a r y gland t h r o u g h o u t these studies. These results permitted us to o b t a i n the m e a s u r e m e n t o f the enzyme in the homogenates. Table 1. Substrate specificity of uterine and mammary gland microsomal fractions Substrate Percentageof enzyme activity (2 raM) Uterus Mammary gland ATP 100.0 100.0 ADP 65.7 77.0 GTP 70.4 76.0 GDP 42.8 60.2 CTP 80.3 77.0 CDP 48.1 56.3 UTP 81.0 71.7 UDP 56.9 55.6 TTP 81.9 62.5 TDP 47.0 54.9 Assay conditions are described in Materials and Methods. All experiments were run at least in duplicate.
115
Apyrase changes in uterus and mammary gland Table 2. Apyras¢ and marker enzymes distribution in fractions obtained after discontinuous sucrose density gradient centrifugation of mammary gland A B C D Mierosomal (8.2/29) (29/38.4) (38.4/50) (50/60) Pellet fraction Interphase lnterphase Interphase Interphase Apyrase: (a) ATPase Specific activity* Purification (times) Recovery (%) (b) ADPase Specific activity Purification (times) Recovery (%) 5'-Nucleotidase Specific activity Purification (times) Recovery (%) Glucose-6-phosphatasc Specific activity x 103 Purification (times) Recovery (%) Galactosyl transferase Specific activity x 103 Purification (times) Recovery (%)
0.19 1.0 100.0
0.42 2.2 8.4
0.42 2.2 7.4
0.20 1.1 1.8
0.05 0.3 0.2
0.06 0.3 1.3
0.16 1.0 100.0
0.35 2.2 8.9
0.34 2.1 8.7
0.16 1.0 4.0
0.03 0.2 0.8
0.03 0.2 0.7
0.08 1.0 100.0
0.21 2.6 10.5
0.21 2.6 9.2
0.10 1.3 2.2
0.03 0.4 0.3
0.17 2.1 1.3
1.08 8.3 14.9
4.41 33.9 33.7
0.05 0.4 2.3
0.54 4.9 9.0
0.02 0.2 1.0
0.13 ' 1.0 100.0
0 0 0
0.11 1.0 100.0
0 0 0
0.46 4.2 17.0
0.57 5.2 18.0
0 0 0
*U/rag protein
Apyrase activity of uterine tissue during pregnancy and involution T h e A T P a s e a n d A D P a s e total units ( c o r r e s p o n d ing to a p y r a s e ) p e r g o f tissue d e c r e a s e d parallelly d u r i n g p r e g n a n c y a n d r e t u r n e d to their initial values at its i n v o l u t i o n (Fig. la). T h e s a m e p a t t e r n o f c h a n g e s w a s o b t a i n e d w h e n d a t a were e x p r e s s e d in t e r m s o f p r o t e i n ( d a t a n o t s h o w n ) o r tissue D N A c o n t e n t (Table 3).
T h e u t e r u s weight increases e i g h t - f o l d as a n average, b u t A T P a s e - A D P a s e activities increase o n l y a b o u t f o u r - f o l d (Table 3). This m e a n s t h a t the total tissue a p y r a s e c o n t e n t increases less t h a n t h e o r g a n size.
Apyrase activity of mammary gland during pregnancy-lactation and involution
160-
A significant c h a n g e in A T P a s e - A D P a s e activities is also o b s e r v e d at the e n d o f p r e g n a n c y (19 days) w h e n b o t h activities begin to increase (Fig. lb). B o t h e n z y m i c activities r e m a i n high d u r i n g l a c t a t i o n a n d after w e a n i n g (8 days). T h e activity expressed in terms o f p r o t e i n o r tissue D N A c o n t e n t also increases, as c a n be seen in T a b l e 4.
12Di
Apyrase activity during the estrous cycle
20D-
a ) uTERus
A T
A T P a s e - A D P a s e activities did n o t c h a n g e d u r i n g t h e e s t r o u s cycle (Table 5). I n spite o f these results t h e rats were i m p l a n t e d w i t h silastic capsules with estradiol w h i c h m a i n t a i n e d estradiol c o n c e n t r a t i o n for 8 days at levels e q u i v a l e n t to t h o s e in p r o e s t r o u s . This t r e a t m e n t causes a light b u t significative increase in b o t h A T P a s e a n d A D P a s e activities (Table 6).
8O
".,~
4.0
to
0
I
i
b) M A . . A R v
i
"/J
i
]~
G-A.O
..,,,~
,o
2.0
,"~""X"'"
b
i
,o
Pregnancy
) /I
l/
,b
20
Lactation
i
,o
Weaning
Days Fig. l. Apyrase activity during the lactogenic cycle ( virginity -pregnancy - lactation -unweaning ) ATPase • • , ADPase O O. The data shown are the mean of five different rats, independently processed, except for virginity which is the mean o f 8 animals.
Table 3. ATPase-ADPase activities in uterus Total units Units per mg of DNA per whole uterus ATPase ADPase ATPase ADPase Virginity (8)* 12.3 __ 1.0t 8.4 -+ 0.6 7.0 _+0.5 5.5 + 0.7 Pregnancy 10 days (5) 9.2+1.8 7.7+2.3 28.8+1.3 17.1+3.2 15 days (5) 5.2 + 1.2 5.2 + 1.1 26.8+ 1.8 20.8+ 1.1 19 days (5) 6.3_+3.2 4.1_+1.9 26.3_+8.1 17.6+3.3 Involution 1 day (5) 16.0 _+0.8 8.8 + 0.5 14.7_+2.7 9.5 + 1.7 8 days (5) 12.2 _+0.9 7.6 +_0.6 4.6 _+0.9 2.3 _+0.3 15 days (5) 15.8 _+3.6 9.2 + 1.2 5.7 _+ 1.4 3.8 + 1.0 *N = number of rats. tSEM. Assay conditions as described in Material and Methods.
M. A. VALENZUELAet al.
116
Table 4. ATPase-ADPase activities in mammary gland Units per mg of D N A ATPase ADPase Virginity (8)* Pregnancy 10 days (5) 15 days (5) 19 days (5) Lactation 4 days (5) 8 days (5) 15 days (5) Involution 8 days (5)
1.54 _+ 0.49~"
1.22 _+ 0.35
1.49 + 0.43 1.94_+0.77 1.50_+0.25
1.32 + 0.22 1.13_+0.37 1.56_+0.13
2.06 _+ 0.66 2.67_+0.19 2.63-+0.66
1.28 _+ 0.27 1.80_+0.09 1.79_+0.40
2.3 -+ 0.43
1.71 + 0.19
*N = Number of rats. tSEM. Assay conditions are described in Materials and Methods.
Platelet antiaggregation activity o f uterine samples
The aggregation response on the addition of ADP is decreased in the presence of uterus homogenates. Figure 2 shows the aggregometer recording of uterine samples obtained from virgin and 19-day-pregnant rats. A clear correlation between the ADPase activity and the reduction in maximal platelet aggregation was observed. The different shape of aggregatory waves obtained when homogenates or microsomal fractions were used probably indicates that the homogenate contained a PGI-like activity in addition to ADPase activity. The characteristic pattern of reversal aggregation seen with ADP-degrading enzymes (Hutton et al., 1980), corresponds to the one observed with the microsomal fraction.
DISCUSSION
The main purpose of this work was to illustrate the approach to the possible roles of apyrase in uterus and mammary gland. These tissues were chosen as models, in periods of high-energy demands, to study the relationship between the enzyme and cell proliferation and differentiation. Apyrase activity changes during the pregnancy-lactation cycle both in uterine tissue and in mammary gland. Experiments are in progress in order to discriminate the changes in the ATPase-ADPase activities, which should be related to its physiological function, and to discover if the changes are associated with kinetic control or with changes in synthesis and/or degradation of apyrase. The kinetic control could be exerted through the modulatory proteins of apyrase that have already been detected (Valenzuela et al., 1989). Uterus from virgin rats has a higher apyrase activity (expressed in units per g of tissue, or per mg of DNA) than the uterus of 19 days of pregnancy. This enzymatic activity directly correlates with the antiaggregatory platelet activity. The increase in total units
of enzyme per whole organ can be attributed to the higher vascularization of the uterus as it increases in size during pregnancy. In support of this suggestion, Miura et al. (1987) have shown the presence of apyrase in vascular walls. Probably, apyrase activity of the uterine tissue could be involved in the inhibition of ADP-induced platelet aggregation (Born, 1962) through the hydrolysis of ADP. Extracellular ADP, released from activated platelets after hemolysis and tissue damage is not only a pro-aggregatory agent, but also promotes thromboxane synthesis. both leading to thrombosis (Gordon, 1986). The further metabolization of extracellular monophosphorylated nucleosides by 5'-nucleotidase produces adenosine. Adenosine, inhibits platelet aggregation, and it is a vasodilator of uterine tissue (Burnstock, 1975). The higher blood supply to uterus during the high energetic demand period studied (pregnancy), could be due to a simultaneous local decrease of ADP and increase in adenosine concentration of the arteriole walls. Uterine smooth muscle presents a high sensitivity to adenine nucleotides acting as neurotransmitters. ATP causes muscle contraction and adenosine produces relaxation (Burnstock, 1975). The changing activity of apyrase could be due to the participation in the regulation of ATP and adenosine levels, which have opposite physiological functions. Uterine apyrase may be regulated by the action of several hormones because of the discordance between the effect of maintained estradiol doses (although the effect is rather small) with the low activity of this enzyme at the end of pregnancy, where the highest uterine levels of this hormone occur. On the other hand, apyrase activity increases in mammary gland during lactation, a period where the gland produces large amounts of lactose and glycosylated proteins, membrane bound and secretable to the milk (Anderson et al, 1982). The level of many enzymes, directly related to the milk components synthesis, increases with respect to the non-secretory tissue (Baldwin and Milligan, 1966), but changes in ATPase ADPase activities have not been followed by any group. Mammary gland apyrase might be involved in protein glycosylation and lactose synthesis because this enzymatic activity is enhanced during lactation. This enzyme could accelerate these processes by drainage of the diphosphate nucleosides and of dolycholpyrophosphate. Protein glycosylation is produced in the rough endoplasmatic reticulum and finished in the Golgi apparatus (Hirsckberg and Snider, 1987). With the purpose of approaching the apyrase physiological role, we studied the subcellular localization of this enzyme in the mammary gland. We found that apyrase co-purified with 5'-nucleotidase, a plasma membrane enzyme marker. Therefore,
Table 5. Apyrase levels in uterus along estral cycle ATPase
Estrous (8)* Diestrous (8) Proestrous (8)
ADPase
U/g tissue
sp. act. U/mg protein
U/g tissue
sp. act. U/mg protein
ATPase ADPase
12.9 + 1.15t 11.2 _ 1.09 12.5 _+ 1.10
0.30+0.022 0.35 ___0.032 0.25 + 0.036
10.5+_1.16 9.4 + 1.25 11.1 + 0.65
0.25_+0.025 0.30 + 0.025 0.23 _+ 0.02
1,16
*N = number of rats. "['SEM.
1.21 1.17
Apyrase changes in uterus and mammary gland
117
Table 6. ATPase--ADPase activities in uterus mantained with estradiol ATPase Total units U/mg DNA (whole organ) 5.30 + 0.80 6.25 _.+0.69 18
Control (8)* Proestrus constant (8) Increase (%)
3.0 _+ 0.25 4.6 + 0.25 53
ADPase Total units U/mg DNA (whole organ) 4.2 +_.0.54 4.9 + 0.5 17
2.6 __.0.20 3.8 + 0.24 46
*N = number of rats. tSEM. Assay conditions are described in Materials and Methods. Constant pro-estrous was sustained by implantation of a silastic capsule with 40 ~ g/ml of 17-fl-estradiol.
a direct function of apyrase in the initial process can be discarded because this enzyme is associated with the plasma membrane and not the endoplasmic reticulum. On the other hand the nucleoside diphosphatase, present in the Golgi apparatus involved in protein glycosylation and lactose synthesis differs from apyrase in its substrate specificity, because it hydrolyzes UDP, G D P and IDP but practically does not hydrolyze adenine nucleotides. There are also differences in the bivalent metal ion stimulation (Novikoff and Goldfischer 1961; Kuhn and White 1977). These results indicate that perhaps apyrase function in the mammary gland might be related to the nucleotide extracellular metabolism. Freshly secreted goat, human and bovine milks contain ATP in different proportions (Zulak et al., 1976). The function of this nucleotide in milk is unknown. Those authors proposed that the amount of ATP or an ATP/ADP ratio may signal reversible inhibition of milk secretion. It is also possible that adenosine could be needed by the newborn to supply a portion of its required purines. It is also possible assign to apyrase the same function proposed by Lalibert6 and Beaudoin (1983) in the pancreas. According to these authors, apyrase
could participate either in the processing of secretory proteins in the condensing vacuoles, or in the exocytosis of the granule content of the pancreatic cell lumen. We assumed that in uterus, apyrase is also bound to plasma membrane, because Matlib et al. (1979) isolated different types of membranes from rat myometrium, and only in the fraction corresponding to plasma membane was an ATPase activity, probably due to apyrase, detected. ATP, A D P (Gordon, 1986) and U D P (Shur, 1989) are nucleotides known to be liberated to the extracellular medium. UDP is released to the external surface of cells as a product of the reaction catalyzed by cell surface glycosyltransferase. This enzyme has been demonstrated to be present in uterine tissue and also in rat mammary tumors (Shur, 1989). Extracellular ATP at low micromolar concentrations can influence processes such as neurotransmission, secretion and membrane permeability (Gordon, 1986). Clearance of ATP from blood may be very important as a regulatory mechanism of the diverse processes already mentioned. The broad specificity towards nucleoside di- and triphosphates and its plasma membrane localization both in uterus and mammary gland, suggest that apyrase, together with 5'-nucleotidase, could be the enzyme responsible for extracellular nucleotide degradation.
o-
Acknowledgements--This w o r k
was financed by F o n d e c y t G r a n t No. 90-1006. W e are grateful to D r C h r i s t o p h e r P o g s o n for his critical reading o f the m a n u s c r i p t .
20-
REFERENCES
40-
fl i
0305-0491/92 $5.00 + 0.00 © 1992Pergamon Press Ltd
CHANGES IN APYRASE ACTIVITY IN UTERUS A N D M A M M A R Y G L A N D D U R I N G THE LACTOGENIC CYCLE M. A. VALENZUELA,L. COLLADOS,A. M. KET'rLUN, M. MANCILLA,H. LARA, J. PUENTE, E. ARANDA,*L. CHAYET,A. ALVAREZ and A. TRAVERSO-CORI Departamento de Bioqulmica y Biologia Molecular, Facultad de Ciencias Qulmicas y Farmac~uticas, Universidad de Chile, Casilla 233-Santiago I, Chile (Fax: 562-2227900); and *Departamento de Hematologia, Facultad de Medicina, Pontificia Universidad Cat61ica, Santiago, Chile (Received 6 February 1992; accepted 6 March 1992)
Abstract--1. The purpose of this present research was to explore the possible roles of ATP-diphosphohydrolase (apyrase) in two tissues with high energetic demands during cell proliferation and differentiation. 2. Changes in apyrase activities during the pregnancy lactation cycle were examined in the rat uterus and mammary gland. 3. A significant decrease in apyrase activity (ATPase-ADPase) was observed in the pregnant uterus; this observation correlates with a minor inhibitory effect on platelet aggregation. 4. In mammary gland, the enzyme activity increases during lactation in parallel with an increase in blood supply, synthesis of glycoproteins and cell proliferation. 5. Apyrase activity did not change during the estrous cycle. Estradiol administration to rats slightly increased (20%) both ATPase-ADPase activities. 6. The probable function of apyrase is finally discussed, based on its substrate specifity and subcellular localization.
INTRODUCTION The presence of an ATPase-ADPase activity corresponding to ATP-diphosphohydrolase (EC 3.6.1.5), or apyrase, has been shown in several animal cells and tissues (Liithje et al., 1988; Valenzuela et al., 1989; Schadeck et al., 1989; Papamarcaki and Tsolas, 1990; Pieber et al., 1991). The enzyme is principally located in the microsomal fraction, and its activity depends on bivalent cations, Ca 2÷ being the most effective. This ATPase-ADPase activity is inhibited neither by oligomycin nor by ouabain, specific inhibitors of mitochondrial and (Na ÷, K+)-ATPase, respectively (LeBel et al., 1980; Knowles et al., 1983; Miura et aL, 1987; Valenzuela et al., 1989, Papamarcaki and Tsolas, 1990). The ability of the enzyme to hydrolyze both ATP and A D P to A M P and inorganic phosphate has suggested a functional role for ATP-diphosphohydrolase in platelet aggregation. This suggestion is based mainly on the extracellular localization of the enzyme in the plasma membrane of the aortic epithelium (Miura et al., 1987; Yagi et al., 1989) and as an ectoenzyme in erythrocytes (Liithje et al., 1988). This possibility is also supported by Hutton et al. (1980) who found that fresh myometrial extracts containing ADPase activity (which, at the present, we know must correspond to apyrase) inhibited platelet aggregation in a concentration-dependent manner. Recently, we have also found a similar effect in a microsomal fraction of rat placenta (Pieber et al., 1991). Due to the high metabolic activity and differentiation of the rat uterus and mammary gland during the pregnancy-lactation cycle, many studies have been focused on ATPase activities present in these
tissues. Although several ATPases have been described in these tissues their relationship to apyrase and its physiological role is unknown (Carraway et al., 1980; Enyedi et aL, 1988; Missaen et al., 1988). Some of the enzymes described are responsible for the ATP-dependent Ca2+-uptake, and a Ca2+/Mg 2+stimulated ATPase has also been identified, but no physiological function for this activity has yet been proposed. The enzyme described in this paper (an apyrase) does not correspond to the ATPase involved in Ca 2+ uptake, because they differ significantly in substrate and bivalent metal ion specificity and in insensitivity to sulfhydryl reagents (Enyedi et al., 1988; Valenzuela et aL, 1989). In the present paper we present evidence of the changes occurring to this enzyme with ATPaseADPase activities in rat uterus and mammary gland during pregnancy-lactation and estrous cycles and the putative role of this enzyme in the physiology of these organs based on its subcellular localization and specificity on nucleoside di- and triphosphates.
MATERIAL AND METHODS
Animals Adult Sprague-Dawley rats (weight 250-200 g) produced in our facilities were housed under controlled conditions of photoperiod (12L: 121)) and temperature (22°C) and were fed with rat chow pellets ad libitum. Estradiol treatment was initiated by subcutaneous implantation of a silastic capsule (20ram in length/100g body weight; o.d, 2.16ram; i.d, 1 ram) containing estradioi dissolved in corn oil at 200#g/ml, a concentration that produces normal serum levels at proestrus (Ojeda et al., 1986).
113
114
M.A. VALENZUELAet al.
Enzyme assay Apyrase activity was followed with ATP (ATPase activity) or ADP (ADPase activity) as substrates (final concentration, 2 mM) in the presence of 5 mM CaCI z in 100 mM Tris HCI, pH 8.0. Pi released was determined by the method of Fiske and SubbaRow (1925). A unit of activity (U) is equivalent to 1 #mol of Pi liberated per min at 30°C. The activity of other nucleoside di- and triphosphate was tested under the same conditions. The true ATPase and ADPase activities corresponding to apyrase were measured in homogenates substracting the possible presence of mitochondrial ATPase, (Na ÷, K +) ATPase and adenylate kinase through the effect of their specific inhibitors. The presence of mitochondrial ATPase in pregnant uterus and lactating mammary gland could be demonstrated by the inhibitory effect of oligomycin (0.9 mg/ml), but the activity was low in comparison to that of apyrase which constituted about 80% of ATPase activity measured. Oligomycin did not affect the activity of the homogenates of both virgin tissues. (Na +, K ÷ )-ATPase was not detected, as indicated by the lack of effect of 5 mM ouabain. The adenylate kinase inhibitor, Ap5A (Feldaus et al., 1975) inhibited apparent ADPase activity by 10% throughout, so the possibility that this activity that this activity arise from a combination of ATPase with adenylate kinase can be discarded.
Protein determination Protein concentrations were measured by the method of Lowry et al. (1951) using BSA as standard.
DNA determination DNA levels were determined according to Burton (1956).
Subcellular fractionation The subcellular fractionation procedure was as previously described (Valenzuela et al., 1989).
Further purification of the microsomal fraction Mammary gland, from rats in pregnancy or lactancy, (between 5 and 6 g) were fractionated to obtain microsomal fraction as already mentioned. The 100,000g pellet was suspended in 2.5 ml of the homogenization buffer. This suspension, containing 8.5% sucrose, 25 mM KC1, 50 mM Tris-HC1, pH 7.5, was placed at the top. of 6 ml of each of the following sucrose layers dissolved in 50 mM Tris-HC1, pH 7.5: 60, 50, 38.7 and 29%. The centrifugation was done in a Beckman Spinco Model L centrifuge with a SW 25 rotor for 60 min at 40,100 g. The fractions of the interfaces 8.2/29, 29/38.7 and 50/60 containing the membrane bands were collected and diluted with distilled water to 8.5% sucrose and centrifuged at 100,000g for 60 min. A small pellet was also obtained at the bottom of the gradient and this fraction and the resulting pellets, after the last centrifugation, were suspended in 0.5 ml of the homogenization buffer. The 5'-nucleotidase and glucose-6-phosphatase, measured as described by Beaufay et al. (1974), were used as plasma membrane and endoplasmic reticulum marker, respectively. The Golgi apparatus was detected through the galactosyl transferase according to Fleisher and Kewina (1974).
Platelet aggregation Platelet aggregation was monitored in an aggregometer PAP-4 (Bio-Data Corp.) using citrate-treated platelet-rich plasma according to Born (Born, 1962). A final concentration of 3/~M of ADP was added to induce aggregation. The decrease in light scattering was followed and results expressed as a percentage of the values found in the controls where tissue samples were replaced by either the homogenization buffer or 0.15 M NaC1.
RESULTS
Substrate specificity o f uterine and mammary microsomal fractions M i c r o s o m a l fractions of b o t h tissues with none of the interfering enzymatic activities (Valenzuela et al., 1989) m e n t i o n e d in Materials a n d M e t h o d s , were used to determine substrate specificity. The fractions of b o t h tissues isolated from virgin rats showed a r a t h e r b r o a d specificity towards nucleosides di- a n d t r i p h o s p h a t e s (Table 1). The higher action o n the t r i p h o s p h a t e derivatives is more i m p o r t a n t with the uterine sample.
Further purification o f microsomal./?actions The different types of m e m b r a n e s of the microsomal pellet were partially separated by discontinuous sucrose gradient. The distribution o f apyrase was n o t different w h e n the gland was isolated from the pregnancy period or from the lactation period. The d a t a s h o w n c o r r e s p o n d to 4 days o f lactation. Purity a n d recovery o f the isolated m e m b r a n e fractions were determined by the use of enzymatic markers. The A T P a s e - A D P a s e activities c o r r e s p o n d i n g to apyrase were f o u n d p r e d o m i n a n t l y at the interfaces 8 . 5 - 2 9 % ( F r a c t i o n A) a n d 2 9 - 3 8 . 7 % ( F r a c t i o n B) of the sucrose gradient. Table 2 shows t h a t the e n r i c h m e n t in specific activities a n d distribution of apyrase parallel the 5' nucleotidase activity. N o glucose-6-phosphatase were detected in these m e m b r a n e fractions, discarding the association o f apyrase to the endoplasmic reticulum. A l t h o u g h these fractions are highly c o n t a m i n a t e d with Golgi apparatus, since galactosyl transferase activity was detected, a nucleoside d i p h o s p h a t a s e different from apyrase has been described in this organelle ( N o v i k o f f a n d Goldfischer, 1961; K u h n a n d White, 1977). The c o n t a m i n a t i o n with other ATPases or adenylate kinase in fractions A a n d B m a y be ruled o u t because apyrase activity of these fractions was u n c h a n g e d when olygomicin, o u a b a i n or A p 5 A were added.
Microsomal distribution o f apyrase during the pregnancy-lactogenie cycle A T P a s e a n d A D P a s e activities showed a microsomal distribution b o t h in uterus a n d m a m m a r y gland t h r o u g h o u t these studies. These results permitted us to o b t a i n the m e a s u r e m e n t o f the enzyme in the homogenates. Table 1. Substrate specificity of uterine and mammary gland microsomal fractions Substrate Percentageof enzyme activity (2 raM) Uterus Mammary gland ATP 100.0 100.0 ADP 65.7 77.0 GTP 70.4 76.0 GDP 42.8 60.2 CTP 80.3 77.0 CDP 48.1 56.3 UTP 81.0 71.7 UDP 56.9 55.6 TTP 81.9 62.5 TDP 47.0 54.9 Assay conditions are described in Materials and Methods. All experiments were run at least in duplicate.
115
Apyrase changes in uterus and mammary gland Table 2. Apyras¢ and marker enzymes distribution in fractions obtained after discontinuous sucrose density gradient centrifugation of mammary gland A B C D Mierosomal (8.2/29) (29/38.4) (38.4/50) (50/60) Pellet fraction Interphase lnterphase Interphase Interphase Apyrase: (a) ATPase Specific activity* Purification (times) Recovery (%) (b) ADPase Specific activity Purification (times) Recovery (%) 5'-Nucleotidase Specific activity Purification (times) Recovery (%) Glucose-6-phosphatasc Specific activity x 103 Purification (times) Recovery (%) Galactosyl transferase Specific activity x 103 Purification (times) Recovery (%)
0.19 1.0 100.0
0.42 2.2 8.4
0.42 2.2 7.4
0.20 1.1 1.8
0.05 0.3 0.2
0.06 0.3 1.3
0.16 1.0 100.0
0.35 2.2 8.9
0.34 2.1 8.7
0.16 1.0 4.0
0.03 0.2 0.8
0.03 0.2 0.7
0.08 1.0 100.0
0.21 2.6 10.5
0.21 2.6 9.2
0.10 1.3 2.2
0.03 0.4 0.3
0.17 2.1 1.3
1.08 8.3 14.9
4.41 33.9 33.7
0.05 0.4 2.3
0.54 4.9 9.0
0.02 0.2 1.0
0.13 ' 1.0 100.0
0 0 0
0.11 1.0 100.0
0 0 0
0.46 4.2 17.0
0.57 5.2 18.0
0 0 0
*U/rag protein
Apyrase activity of uterine tissue during pregnancy and involution T h e A T P a s e a n d A D P a s e total units ( c o r r e s p o n d ing to a p y r a s e ) p e r g o f tissue d e c r e a s e d parallelly d u r i n g p r e g n a n c y a n d r e t u r n e d to their initial values at its i n v o l u t i o n (Fig. la). T h e s a m e p a t t e r n o f c h a n g e s w a s o b t a i n e d w h e n d a t a were e x p r e s s e d in t e r m s o f p r o t e i n ( d a t a n o t s h o w n ) o r tissue D N A c o n t e n t (Table 3).
T h e u t e r u s weight increases e i g h t - f o l d as a n average, b u t A T P a s e - A D P a s e activities increase o n l y a b o u t f o u r - f o l d (Table 3). This m e a n s t h a t the total tissue a p y r a s e c o n t e n t increases less t h a n t h e o r g a n size.
Apyrase activity of mammary gland during pregnancy-lactation and involution
160-
A significant c h a n g e in A T P a s e - A D P a s e activities is also o b s e r v e d at the e n d o f p r e g n a n c y (19 days) w h e n b o t h activities begin to increase (Fig. lb). B o t h e n z y m i c activities r e m a i n high d u r i n g l a c t a t i o n a n d after w e a n i n g (8 days). T h e activity expressed in terms o f p r o t e i n o r tissue D N A c o n t e n t also increases, as c a n be seen in T a b l e 4.
12Di
Apyrase activity during the estrous cycle
20D-
a ) uTERus
A T
A T P a s e - A D P a s e activities did n o t c h a n g e d u r i n g t h e e s t r o u s cycle (Table 5). I n spite o f these results t h e rats were i m p l a n t e d w i t h silastic capsules with estradiol w h i c h m a i n t a i n e d estradiol c o n c e n t r a t i o n for 8 days at levels e q u i v a l e n t to t h o s e in p r o e s t r o u s . This t r e a t m e n t causes a light b u t significative increase in b o t h A T P a s e a n d A D P a s e activities (Table 6).
8O
".,~
4.0
to
0
I
i
b) M A . . A R v
i
"/J
i
]~
G-A.O
..,,,~
,o
2.0
,"~""X"'"
b
i
,o
Pregnancy
) /I
l/
,b
20
Lactation
i
,o
Weaning
Days Fig. l. Apyrase activity during the lactogenic cycle ( virginity -pregnancy - lactation -unweaning ) ATPase • • , ADPase O O. The data shown are the mean of five different rats, independently processed, except for virginity which is the mean o f 8 animals.
Table 3. ATPase-ADPase activities in uterus Total units Units per mg of DNA per whole uterus ATPase ADPase ATPase ADPase Virginity (8)* 12.3 __ 1.0t 8.4 -+ 0.6 7.0 _+0.5 5.5 + 0.7 Pregnancy 10 days (5) 9.2+1.8 7.7+2.3 28.8+1.3 17.1+3.2 15 days (5) 5.2 + 1.2 5.2 + 1.1 26.8+ 1.8 20.8+ 1.1 19 days (5) 6.3_+3.2 4.1_+1.9 26.3_+8.1 17.6+3.3 Involution 1 day (5) 16.0 _+0.8 8.8 + 0.5 14.7_+2.7 9.5 + 1.7 8 days (5) 12.2 _+0.9 7.6 +_0.6 4.6 _+0.9 2.3 _+0.3 15 days (5) 15.8 _+3.6 9.2 + 1.2 5.7 _+ 1.4 3.8 + 1.0 *N = number of rats. tSEM. Assay conditions as described in Material and Methods.
M. A. VALENZUELAet al.
116
Table 4. ATPase-ADPase activities in mammary gland Units per mg of D N A ATPase ADPase Virginity (8)* Pregnancy 10 days (5) 15 days (5) 19 days (5) Lactation 4 days (5) 8 days (5) 15 days (5) Involution 8 days (5)
1.54 _+ 0.49~"
1.22 _+ 0.35
1.49 + 0.43 1.94_+0.77 1.50_+0.25
1.32 + 0.22 1.13_+0.37 1.56_+0.13
2.06 _+ 0.66 2.67_+0.19 2.63-+0.66
1.28 _+ 0.27 1.80_+0.09 1.79_+0.40
2.3 -+ 0.43
1.71 + 0.19
*N = Number of rats. tSEM. Assay conditions are described in Materials and Methods.
Platelet antiaggregation activity o f uterine samples
The aggregation response on the addition of ADP is decreased in the presence of uterus homogenates. Figure 2 shows the aggregometer recording of uterine samples obtained from virgin and 19-day-pregnant rats. A clear correlation between the ADPase activity and the reduction in maximal platelet aggregation was observed. The different shape of aggregatory waves obtained when homogenates or microsomal fractions were used probably indicates that the homogenate contained a PGI-like activity in addition to ADPase activity. The characteristic pattern of reversal aggregation seen with ADP-degrading enzymes (Hutton et al., 1980), corresponds to the one observed with the microsomal fraction.
DISCUSSION
The main purpose of this work was to illustrate the approach to the possible roles of apyrase in uterus and mammary gland. These tissues were chosen as models, in periods of high-energy demands, to study the relationship between the enzyme and cell proliferation and differentiation. Apyrase activity changes during the pregnancy-lactation cycle both in uterine tissue and in mammary gland. Experiments are in progress in order to discriminate the changes in the ATPase-ADPase activities, which should be related to its physiological function, and to discover if the changes are associated with kinetic control or with changes in synthesis and/or degradation of apyrase. The kinetic control could be exerted through the modulatory proteins of apyrase that have already been detected (Valenzuela et al., 1989). Uterus from virgin rats has a higher apyrase activity (expressed in units per g of tissue, or per mg of DNA) than the uterus of 19 days of pregnancy. This enzymatic activity directly correlates with the antiaggregatory platelet activity. The increase in total units
of enzyme per whole organ can be attributed to the higher vascularization of the uterus as it increases in size during pregnancy. In support of this suggestion, Miura et al. (1987) have shown the presence of apyrase in vascular walls. Probably, apyrase activity of the uterine tissue could be involved in the inhibition of ADP-induced platelet aggregation (Born, 1962) through the hydrolysis of ADP. Extracellular ADP, released from activated platelets after hemolysis and tissue damage is not only a pro-aggregatory agent, but also promotes thromboxane synthesis. both leading to thrombosis (Gordon, 1986). The further metabolization of extracellular monophosphorylated nucleosides by 5'-nucleotidase produces adenosine. Adenosine, inhibits platelet aggregation, and it is a vasodilator of uterine tissue (Burnstock, 1975). The higher blood supply to uterus during the high energetic demand period studied (pregnancy), could be due to a simultaneous local decrease of ADP and increase in adenosine concentration of the arteriole walls. Uterine smooth muscle presents a high sensitivity to adenine nucleotides acting as neurotransmitters. ATP causes muscle contraction and adenosine produces relaxation (Burnstock, 1975). The changing activity of apyrase could be due to the participation in the regulation of ATP and adenosine levels, which have opposite physiological functions. Uterine apyrase may be regulated by the action of several hormones because of the discordance between the effect of maintained estradiol doses (although the effect is rather small) with the low activity of this enzyme at the end of pregnancy, where the highest uterine levels of this hormone occur. On the other hand, apyrase activity increases in mammary gland during lactation, a period where the gland produces large amounts of lactose and glycosylated proteins, membrane bound and secretable to the milk (Anderson et al, 1982). The level of many enzymes, directly related to the milk components synthesis, increases with respect to the non-secretory tissue (Baldwin and Milligan, 1966), but changes in ATPase ADPase activities have not been followed by any group. Mammary gland apyrase might be involved in protein glycosylation and lactose synthesis because this enzymatic activity is enhanced during lactation. This enzyme could accelerate these processes by drainage of the diphosphate nucleosides and of dolycholpyrophosphate. Protein glycosylation is produced in the rough endoplasmatic reticulum and finished in the Golgi apparatus (Hirsckberg and Snider, 1987). With the purpose of approaching the apyrase physiological role, we studied the subcellular localization of this enzyme in the mammary gland. We found that apyrase co-purified with 5'-nucleotidase, a plasma membrane enzyme marker. Therefore,
Table 5. Apyrase levels in uterus along estral cycle ATPase
Estrous (8)* Diestrous (8) Proestrous (8)
ADPase
U/g tissue
sp. act. U/mg protein
U/g tissue
sp. act. U/mg protein
ATPase ADPase
12.9 + 1.15t 11.2 _ 1.09 12.5 _+ 1.10
0.30+0.022 0.35 ___0.032 0.25 + 0.036
10.5+_1.16 9.4 + 1.25 11.1 + 0.65
0.25_+0.025 0.30 + 0.025 0.23 _+ 0.02
1,16
*N = number of rats. "['SEM.
1.21 1.17
Apyrase changes in uterus and mammary gland
117
Table 6. ATPase--ADPase activities in uterus mantained with estradiol ATPase Total units U/mg DNA (whole organ) 5.30 + 0.80 6.25 _.+0.69 18
Control (8)* Proestrus constant (8) Increase (%)
3.0 _+ 0.25 4.6 + 0.25 53
ADPase Total units U/mg DNA (whole organ) 4.2 +_.0.54 4.9 + 0.5 17
2.6 __.0.20 3.8 + 0.24 46
*N = number of rats. tSEM. Assay conditions are described in Materials and Methods. Constant pro-estrous was sustained by implantation of a silastic capsule with 40 ~ g/ml of 17-fl-estradiol.
a direct function of apyrase in the initial process can be discarded because this enzyme is associated with the plasma membrane and not the endoplasmic reticulum. On the other hand the nucleoside diphosphatase, present in the Golgi apparatus involved in protein glycosylation and lactose synthesis differs from apyrase in its substrate specificity, because it hydrolyzes UDP, G D P and IDP but practically does not hydrolyze adenine nucleotides. There are also differences in the bivalent metal ion stimulation (Novikoff and Goldfischer 1961; Kuhn and White 1977). These results indicate that perhaps apyrase function in the mammary gland might be related to the nucleotide extracellular metabolism. Freshly secreted goat, human and bovine milks contain ATP in different proportions (Zulak et al., 1976). The function of this nucleotide in milk is unknown. Those authors proposed that the amount of ATP or an ATP/ADP ratio may signal reversible inhibition of milk secretion. It is also possible that adenosine could be needed by the newborn to supply a portion of its required purines. It is also possible assign to apyrase the same function proposed by Lalibert6 and Beaudoin (1983) in the pancreas. According to these authors, apyrase
could participate either in the processing of secretory proteins in the condensing vacuoles, or in the exocytosis of the granule content of the pancreatic cell lumen. We assumed that in uterus, apyrase is also bound to plasma membrane, because Matlib et al. (1979) isolated different types of membranes from rat myometrium, and only in the fraction corresponding to plasma membane was an ATPase activity, probably due to apyrase, detected. ATP, A D P (Gordon, 1986) and U D P (Shur, 1989) are nucleotides known to be liberated to the extracellular medium. UDP is released to the external surface of cells as a product of the reaction catalyzed by cell surface glycosyltransferase. This enzyme has been demonstrated to be present in uterine tissue and also in rat mammary tumors (Shur, 1989). Extracellular ATP at low micromolar concentrations can influence processes such as neurotransmission, secretion and membrane permeability (Gordon, 1986). Clearance of ATP from blood may be very important as a regulatory mechanism of the diverse processes already mentioned. The broad specificity towards nucleoside di- and triphosphates and its plasma membrane localization both in uterus and mammary gland, suggest that apyrase, together with 5'-nucleotidase, could be the enzyme responsible for extracellular nucleotide degradation.
o-
Acknowledgements--This w o r k
was financed by F o n d e c y t G r a n t No. 90-1006. W e are grateful to D r C h r i s t o p h e r P o g s o n for his critical reading o f the m a n u s c r i p t .
20-
REFERENCES
40-
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