Implication of ATP and Sodium in Arachidonic Acid Incorporation by Placental Syncytiotrophoblast Brush Border and Basal Plasma Membranes in the Human

Placenta (2000), 21, 661–669 doi:10.1053/plac.2000.0561, available online at http://www.idealibrary.com on

Implication of ATP and Sodium in Arachidonic Acid Incorporation by Placental Syncytiotrophoblast Brush Border and Basal Plasma Membranes in the Human J. Lafonda,b,c, F. Moukdara,b, A. Riouxa,b, H. Ech-Chadlia,b, L. Brissetteb, J. Robidouxa,b, A. Massed and L. Simoneaua,b a

Laboratoire de Physiologie materno-fœtale, b De´partement des Sciences Biologiques, Universite´ du Que´bec a` Montre´al, Montre´al and d Obstetrics and Gynecology Services, Centre Hospitalier Universitaire de Montre´al, Pavillon St-Luc, Que´bec, Canada, H3C 3P8 Paper accepted 4 April 2000

The human placental syncytiotrophoblast is the main site of exchange of nutrients and minerals between the mother and her fetus. In order to characterize the placental transport of some fatty acids, we studied the incorporation of arachidonic acid, a fetal primordial fatty acid, in purified bipolar syncytiotrophoblast brush border (BBM) and basal plasma membranes (BPM) from human placenta. The basal arachidonic acid incorporation in BBM and BPM was time dependent and reached maximal values of 0.750.10 and 0.480.18 pmol/mg protein, respectively, after 2.5 min. The presence of adenosine triphosphate (ATP) (3 m) increases significantly the maximal incorporation of arachidonic acid by sixfold (4.750.35 pmol/mg) and ninefold (4.400.84 pmol/mg) in BBM and BPM, respectively. Moreover, an increase in the arachidonic acid incorporation was also obtained in the presence of sodium where the values achieved 7.680.98 (10) and 6.53 pmol/mg (13.6) for BBM and BPM, respectively. We also showed that the combination of both Na + and ATP increases significantly the maximal incorporation of arachidonic acid in BPM to 7.890.15 pmol/mg protein, while in BBM it did not modify its incorporation (8.180.25 pmol/mg protein), as compared to the presence of sodium alone. Our results demonstrate that arachidonic acid is incorporated by both placental syncytiotrophoblast membranes, and is ATP and sodium-linked. However, different mechanisms seem to be involved in this fatty acid incorporation through BBM and BPM, since the presence of Na + or ATP increased it, while the association of these two elements increased it only in BPM. We also demonstrated by osmolarity experiments that both membranes bind arachidonic acid, potentially involving one or more fatty acids binding proteins.  2000 Harcourt Publishers Ltd Placenta (2000), 21, 661–669

INTRODUCTION Fetal development and homeostasis until birth depend on the maternal–fetal exchanges of nutrients by the placenta. During the last trimester of pregnancy, essential and non-essential fatty acids are very important for the growth and development of the nervous system, the formation of cellular membranes, and many essential fetal physiological processes (Neuringer et al., 1986; Crawford et al., 1986; Bourre et al., 1990; Holman, Johnson and Ogburn, 1991). The placenta produces, modifies and transfers to the fetus all the non-essential fatty acids it c To whom correspondence should be addressed at: Laboratoire de Physiologie materno-foetale, Universite´ du Que´bec a` Montre´al, De´partement des Sciences Biologiques, C.P. 8888, Succursale ‘‘Centre-Ville’’, Montre´al, Que´bec (CANADA) H3C 3P8. Fax: +1 514 987 4647; E-mail: [email protected]

0143–4004/00/070661+09 $35.00/0

needs (Hummel et al., 1976; Coleman, 1986), while the essential fatty acids are provided to the fetus by the maternal circulation, through the placenta, because the metabolic enzymes required for their synthesis are absent in mammal fetuses (Coleman, 1989; Bourre et al., 1984). In sheep, Shand and Noble (1979) demonstrated a strong accumulation of arachidonic acid in placental and fetal units. However, this accumulation could be caused by the conversion of linoleic acid to arachidonic acid by
6-desaturase activity rather than by an elevated transport in the placenta. Arachidonic acid is known to be an essential fatty acid for the fetus, since it possesses, in sheep, a weak
6-desaturase enzymatic activity (Noble, 1979), but human fetus has to depend on maternal supply of long chain polyunsaturated fatty acid (LCPUFA) (Haggarty et al., 1997). Thus, fetal arachidonic acid should be provided by the female or placental linoleic acid conversion. Arachidonic  2000 Harcourt Publishers Ltd

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acid is involved in cell membrane biogenesis, particularly in the kidney, brain and spinal cord (Bojesen and Bojesen, 1995), and represents a large portion of the syncytiotrophoblast lipids (Lafond, Ayotte and Brunette, 1993). Unfortunately, only a few studies have focused on fatty acid incorporation by human syncytiotrophoblast membranes or the fatty acid transfer towards the fetal circulation. However, a study demonstrating specific linoleic acid incorporation mechanisms, on syncytiotrophoblast membranes of human placenta (Lafond et al., 1994a), shows that this fatty acid is preferentially incorporated by brush border (BBM) and, at a lower rate, by basal plasma membranes (BPM). The transplacental fatty acid exchange is carried out through the bipolar syncytiotrophoblast, which constitutes two types of membranes: a BBM, facing the maternal circulation, and a BPM, facing the fetal circulation. These two membranes are functionally and structurally different (Lafond et al., 1988, 1994a,b; Lafond, Leclerc and Brunette, 1991; Amane et al., 1995; El Mabrouk et al., 1996; Eaton and Oakey, 1997). Moreover, it is well known that placental fatty acid transport is affected by maternal diet and is particularly elevated during the last trimester of pregnancy (Coleman, 1989). Effectively, the human placenta transports lipids at a very rapid rate and produces the fattest of all newborns among many species. At term, lipid stores account for approximately 18 per cent of the total body weight (Widdowson, 1974; Battaglia and Meschia, 1986). Some authors have shown that human placenta preferentially transport the LCPUFA (Lafond et al., 1994a; Campbell, Gordon and Dutta-Roy, 1994, 1996), but the mechanisms involved in this transport process need to be elucidated. These mechanisms, in many tissues, have often been related to passive diffusion (Samuel, Paris and Ailhaud, 1976; Stremmel, Strohmeyer and Brek, 1986). However, many observations suggested that a portion of the LCPUFA uptake is mediated by facilitated transport systems. Recently, Campbell et al. (1994, 1995a,b) showed the presence of a plasma membrane fatty acid binding protein (FABPpm) in human placenta localized exclusively in BBM, while Das, Gaurisankar and Mukherjea (1993) reported finding cytosolic FABP (liver and cardiac types) in human placenta. Also, Campbell et al. (1998) showed the presence of fatty acid translocase (FAT, or CD36) and a fatty acid transport protein (FATP) in both microvillous and basal membranes of human placenta (Campbell et al., 1998). These proteins are localized in many tissues and their concentration varies considerably with the concentration of intra- and extracell lipids. These membraneassociated proteins could play an important role in FFA incorporation, but they also could be only involved in the protection of the vital processes of the cell, assuring a normal maternal–fetal relationship. In the present paper, we investigated the in vitro incorporation of arachidonic acid by BBM and BPM vesicles from syncytiotrophoblast of normal human term placenta, and determined the influence of sodium and ATP on the kinetic parameters of this incorporation.

MATERIALS AND METHODS Materials [3H]-Arachidonic acid was obtained from Amersham Canada (Montreal, Canada). Albumin (free fatty acid) was purchased from Roche Molecular Biochemicals (Laval, Canada) and phloretin from ICN Pharmaceuticals Canada (Montreal, Canada). The Multiscreen rapid filtration system and the 0.65 m polyvinylidene fluoride filters were purchased from Millipore Canada Inc. (Montreal, Canada). All other products were purchased from Sigma Co. (St Louis, MO, USA). Purification of placental membranes Human placenta was obtained from full term normal vaginal deliveries. The amnion, chorion and decidua were removed and the placental tissue (120–150 g) was obtained from the central part of the placenta. The tissue was cut into 2–5 mm fragments and stirred in Tris-HEPES (10 m)-mannitol buffer [10 m Tris-HEPES (pH 7.4) and 270 m mannitol], for 45 min at 4C. After this agitation, BBM and BPM were prepared using the technique of Smith et al. (1974) and modified by Lafond et al. (1988) and Robidoux et al. (1998). Brush border membrane preparation. The homogenate was filtered through cotton gauze, the filtrate was used to prepare the BBM and the residual tissue was used to prepare the BPM. The filtrate obtained was centrifuged for 15 min at 2900 g and the supernatant was centrifuged again at 150 000 g for 60 min. The pellet was suspended in a Tris-HEPES (2 m)-mannitol buffer [2 m Tris-HEPES (pH 7.0) and 270 m mannitol] and stirred for 20 min in the presence of 10 m MgCl2. The mixture was centrifuged for 20 min at 3600 g. The supernatant was centrifuged twice at 35 000 g for 45 min in the TrisHEPES (10 m)-mannitol buffer (pH 7.4), and the purified membranes were stored at 4C until used. Basal plasma membrane preparation. The residual placental tissue obtained after the first agitation was washed with Tris-HEPES buffer [10 m Tris-HEPES (pH 7.4)] and stirred for 45 min with Tris-HEPES (10 m)-mannitol buffer containing 10 m EDTA. The homogenate was filtered through cotton gauze. The filtrate was centrifuged for 15 min at 2900 g, and the supernatant was centrifuged at 150 000 g for 60 min. The pellet was suspended in Tris-HEPES (2 m)mannitol buffer and stirred for 20 min in the presence of 10 m MgCl2. The mixture was centrifuged for 20 min, at 2900 g. The pellet was suspended in Tris-HEPES buffer and stored at
80C for at least 30 min. The suspension was then centrifuged for 30 min at 90 000 g. The pellet was suspended in Tris-HEPES buffer and loaded on top of a discontinuous Ficoll gradient (4 and 10 per cent), and centrifuged for 60 min at 90 000 g. The membranes were collected at the 4–10 per cent interface and centrifuged twice at 35 000 g for 30 min in Tris-HEPES (10 m)-mannitol buffer. The purified membranes were stored at 4C until used.

Lafond et al.: ATP and Sodium Incorporation in Arachidonic Acid

The purity of both membranes was monitored by the measurement of the alkaline phosphatase activity (a BBM marker) as described in Kelly and Hamilton (1970) and by the measurement of the Na + /K + -ATPase activity (a BPM marker) using the technique of Post and Sen (1967). The specificity of these enzyme markers for both membranes have been largely reported (Lafond, Leclerc and Brunette, 1991; Lafond, Ayotte and Brunette, 1993; Lafond et al., 1994a,b). The protein concentrations were determined by the method of Bradford (1976).

ARACHIDONIC ACID INCORPORATION Placental membranes were vesiculated in accordance to the procedure of Lafond et al. (1994a) and Shennan and Boyd (1987), using a tuberculin syringe with a needle gauge of 26G. Purified syncytiotrophoblast BBM and BPM were washed with transport buffer (155 m KCl, 3 m MgCl2, 60 m sucrose, 10 m CaCl2 and 20 m K2HPO4, pH 7.4) and vesiculated in this buffer. Twenty micrograms (50 l) of purified vesiculated syncytiotrophoblast BBM or BPM were added to start the reaction in 200 l of transport buffer containing various concentration of [3H]-arachidonic acid (230.5 Ci/mmol) (0–250 n). Incubations were performed at 37C for different times (0–5 min) and the incorporation process was stopped by the addition of 1 ml of stopping and washing buffer (150 m NaCl, 3 m MgCl2, 5 m KCl, 60 m sucrose, 200  phloretin, 0.1 per cent albumin and 20 m Na2HPO4, pH 7.4). After that, vesicles were collected on filters by rapid filtration using the Multiscreen system (Millipore) with 0.65 m polyvinylidene fluoride filters presoaked with 0.1 per cent albumin as described in Lafond et al. (1994a), and then washed twice with the washing buffer. Radioactivity was determined by liquid scintillation counting using a system 1400 Wallac. In all experiments, the [3H]-arachidonic acid was bound to albumin in a 6 : 1 molar ratio by sonicating the mixture, three times for 2 min, as described in Reuter, Prairie and Kaneshiro (1993). In the experiments regarding the effect of ATP, 3 m of ATP in the form of Na + salt (ATPNa+ ) or 3 m of ATP in the form of Ca2+ salt (ATPCa2+ ) and an ATP regenerating system containing 3 m of phosphocreatine and 100 g/ml of creatine kinase were added to the transport buffer and the membranes were vesiculated. Thus, the ATP and the regenerating system are on both sides of the vesicles, as described in Marin et al. (1995) and Lafond et al. (1994a). Prior to the addition of various concentrations of [3H]-arachidonic acid, membranes were preincubated for 3 min at 37C in presence of ATP and, later on, the incubations were performed at 37C for 150 sec. In the experiments regarding the effects of the Na + , we replaced 155 m of KCl and 20 m of K2HPO4 in the transport buffer, by 150 m of NaCl, 5 m KCl and 20 m Na2HPO4, as described in Marin et al. (1990, 1995). The NaCl (150 m) was added at the same time as the [3H]-arachidonic

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Table 1. Enzymatic parameters of syncytiotrophoblast (alkaline phosphatase and Na + /K + -ATPase) BBM and BPM of human placenta [the purity of both membranes was monitored by measuring alkaline phosphatase (EC 3.1.3.1) (BBM marker) activity and Na + / K + -ATPase (EC 3.6.1.3) (BPM marker)] Fraction

Alkaline phosphatase (mol/mg prot/15 min)

Na + /K + -ATPase (mol/mg prot/min)

Homogenate BBM BPM

7.190.87 194.118.6 (27) 34.005.00 (4)

0.130.03 0.740.30 (3.5) 3.380.37 (26)

a Values of enzyme markers are expressed as the means of five different placentae and the magnitude of enrichment is indicated in parentheses. b P<0.001 compared to homogenate and BBM values.

acid (various concentrations) and the incubations were performed at 37C for 150 sec. In the experiments regarding the effects of Na + and ATP conjointly, the syncytiotrophoblast BBM and BPM were vesiculated in the buffer containing ATP as described earlier and the Na + (150 m) was added at the same time as the [3H]-arachidonic acid and the incubations were performed at 37C for 150 sec. In order to evaluate the quantity of arachidonic acid bound to BBM and BPM, the effect of osmolarity on the arachidonic acid incorporation was also studied. Membranes were vesiculated in transport buffer containing 100 m sucrose. They were pre-incubated at 37C for 3 min, in the presence of increasing external concentrations of sucrose (0 to 3.2 ) allowing vesicles shrinkage. The arachidonic acid incorporation was then performed as previously described. The binding fraction was subtracted from each incorporation value.

Statistical analysis Statistical analysis were performed using the unpaired t-test. Results were expressed as the mean. A level of P<0.05 was considered as significant.

RESULTS Membrane purity Membrane purity was evaluated by specific enzyme markers for both BBM (alkaline phosphatase) and BPM (Na + /K + ATPase). The enrichment values of BBM and BPM are shown in Table 1. The enrichment of alkaline phosphatase was 27-fold and fourfold in BBM and BPM respectively, compared with homogenate. The BPM marker, Na + /K + -ATPase, was enriched 3.5-fold and 26-fold, for BBM and BPM, respectively. Contamination by intracellular organites was very low as previously reported by our research group (Lafond, Leclerc and Brunette, 1991).

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1.5

1.0

0.5

0

100

200 Time (sec)

300

400

Figure 1. Time course of [3H]-arachidonic acid (20 n) incorporation by BBM and BPM. The fatty acid was bound to albumin in a 6 : 1 molar ratio. The experiment was performed with 20 g proteins of BBM at 37C ( ) or BPM at 37C () in a transport buffer (155 m KCl, 3 m MgCl2, 60 m sucrose, 10 m CaCl2 and 20 m K2HPO4, pH 7.4). Results express the mean of three separate experiments in which triplicate determinations were done.

Arachidonic acid incorporation Figure 1 shows the effect of various incubation times on basal arachidonic acid incorporation through BBM and BPM at 37C. The experiments were carried out in the presence of 20 n [3H]-arachidonic acid bound to albumin in a 6 : 1 molar ratio, since the maximal arachidonic acid incorporation was obtained with this ratio (data not shown). The incorporation by these two membranes is characterized by a latency phase up to 20–40 sec to reach about 0.15 pmol/mg protein, followed by a rapid increase to reach the maximal incorporation values of 1.30.08 pmol/mg protein for BBM and 1.40.1 pmol/mg protein for BPM, which are obtained after 180 sec. The use of a cold stopping and washing buffer containing 200  phloretin/0.1 per cent BSA removed non-specific surface bound [3H]-arachidonic acid while blocking efflux of fatty acid from the vesiculated BBM and BPM already internalized, permitting accurate measurement of arachidonic acid incorporation and specific binding (Stremmel, Kachwa and Berk, 1983). The saturation curve of arachidonic acid incorporation by BBM and BPM is shown in Figure 2. Arachidonic acid incorporation shows a saturable mechanism with maximal values reaching 2.91.3 pmol/mg protein for BBM and 3.21.4 pmol/mg protein for BPM in presence of 250 n [3H]-arachidonic acid, for 150 sec at 37C. These results indicate that arachidonic acid incorporation is similar in both syncytiotrophoblast membranes from human placenta. The kinetic parameters of this fatty acid incorporation was analysed using a Lineweaver–Burk transformation and were based on the total arachidonic acid concentrations, which included both the albumin bound and free arachidonic acid. The corresponding plot (insert of Figure 2) shows apparent Km of 22.57.2 and 21.78.0 n, and Vmax values of 0.90.1 and

4 3

1/V (mg/pmol)

2.0 Arachidonic acid incorporation (pmol/mg)

Arachidonic acid incorporation (pmol/mg)

664 3 2 1 0 –0.10 0.00 0.10 0.20 1/S (nM–1)

2 1

0

50

100 200 150 [Arachidonic acid] (nM)

250

Figure 2. [3H]-Arachidonic acid incorporation into BBM and BPM. The fatty acid was bound to albumin in a 6 : 1 molar ratio. The experiment was performed in the presence of increasing concentrations of [3H]-arachidonic acid (0–250 n) and 20 g proteins of BBM ( ) or BPM ( ) at 37C for 150 sec. The insert shows the Lineweaver–Burk plot. Results express the mean of three separate experiments in which triplicate determinations were done.

1.00.2 pmol/mg/min for BBM and BPM, respectively. These data show that the affinity of the carrier for arachidonic acid in BBM and BPM was similar. In accordance, the Vmax/Km values are not significantly different between both syncytiotrophoblast membranes (Table 2). + ) The presence of ATP in the form of Na + salt (ATPNa + (3 m) (Figure 3) or Na (150 m) (Figure 4) significantly increases the maximal incorporation of arachidonic acid in both syncytiotrophoblast membranes as compared to the basal incorporation (without ATP or sodium). The Lineweaver– Burk transformation of these data (inserts of Figures 3 and 4) shows that the Km values for BBM and BPM were not significantly different from the control (without ATP or Na + ) or each other (Table 2). However, Vmax values in the presence + of ATPNa nor Na + were significantly different from the basal incorporation, and were significantly different from each other for both syncytiotrophoblast membranes (Table 2). Moreover, in Table 3, we demonstrated that in BPM, the presence of + , increases significantly the Na + used conjointly with ATPNa maximal incorporation of arachidonic acid (7.890.15 pmol/ + mg) as compared to the medium containing only ATPNa + (4.400.84 pmol/mg) or Na (6.530.20 pmol/mg). How+ was not additive when ever, the effect of Na + and ATPNa both elements were combined. Moreover, in BBM, the maximal incorporation was obtained in the presence of Na + only (Table 3). + ), Since the ATP used was in the form of a Na + salt (ATPNa we also carried out incorporation studies by BBM in the presence of an ATP in the form of a Ca2+ salt (ATP2+ Ca ), in order to discriminate whether the increase of arachidonic acid incorporation observed in the syncytiotrophoblastic membranes is related to the presence of the Na + linked to the ATP or to the ATP only. Results showed that any differences in the arachidonic acid incorporation in BBM were observed in the

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Table 2. Kinetic parameters of arachidonic acid incorporation in syncytiotrophoblast BBM and BPM of normal term human placenta. The fatty acid was bound to albumin in a 6 : 1 molar ratio. The experiment were performed with 20 g proteins of BBM or BPM, for 150 sec at 37C. Results are expressed as the mean in which triplicate determinations of three placentae were performed Membranes

Vmax (pmol/mg prot/min)

Km (n)

Vmax/Km

BBM (basal) BBM with 3 m ATP BBM with 150 m Na + BPM (basal) BPM with 3 m ATP BPM with 150 m Na +

0.90.1 4.30.5a 8.51.0a,b 1.00.2 4.60.5a 8.31.0a,b

22.57.2 8.63.0 16.04.3 21.78.0 11.11.2 15.44.4

0.04 0.5 0.5 0.05 0.4 0.4

P<0.05 versus basal incorporation; bP<0.01 versus ATP.

17.5 15.0

40 2

Arachidonic acid incorporation (pmol/mg)

20.0

1/V (mg/pmol)

Arachidonic acid incorporation (pmol/mg)

3

1 0 –0.1

12.5

0.0 0.1 0.2 1/S (nM–1)

10.0 7.5 5.0

30

1/V (mg/pmol)

a

3 2 1

0 –0.10 0.00 0.10 0.20 1/S (nM–1)

20

10

2.5 0 0

50

150 100 200 [Arachidonic acid] (nM)

250

300

Figure 3. Effect of adenosine triphosphate (ATP) (3 m) on arachidonic acid incorporation into BBM and BPM. Membranes were preincubated 180 sec at 37C, after that the [3H]-arachidonic acid (0–250 n) was added to start the reaction. 3 m ATP and the ATP regenerating system (3 m phosphocreatine and 100 g/ml creatine kinase) were added in the vesiculated medium. The fatty acid was bound to albumin in a 6 : 1 molar ratio. The experiment was performed with 20 g proteins of BBM in absence () or presence ( ) of ATP or with BPM in absence ( ) or presence () of ATP, at 37C during 150 sec. The insert shows the Lineweaver–Burk plot. Results express the means of three separate experiments in which triplicate determinations were done.

presence of ATP in the form of Na + salt (4.750.35 pmol/ mg) or Ca2+ salt (5.200.40 pmol/mg), and the effect of ATP was maintained (Table 3). Moreover, in the presence of Na + combined with ATP, the arachidonic acid incorporation was not modified by the ATPs used. To evaluate the effect of this binding on both syncytiotrophoblast membranes, we verified the effect of an extravesicular osmolarity variation on arachidonic acid incorporation, using an increasing concentration of sucrose, an agent that does not permeate vesicles but has an osmotic effect (Figure 5). Under the lowest extravesicular concentration of sucrose (iso-osmolar condition, 100 m), the arachidonic acid incorporation by BBM and BPM reached about 1.280.07 pmol/mg protein

100 200 [Arachidonic acid] (nM)

300

Figure 4. Effect of a sodium (Na + ) (155 m) on arachidonic acid incorporation into BBM and BPM. Membranes were vesiculated in the transport buffer containing no Na + . The [3H]-arachidonic acid (0–250 n) was added in the modified transport medium, where K + was replaced by Na + . The fatty acid was bound to albumin in a 6 : 1 molar ratio. The experiment was performed with 20 g proteins of BBM in absence () or presence ( ) of Na + or with BPM in absence ( ) or presence () of Na + , at 37C during 150 sec. The insert shows the Lineweaver–Burk plot. Results express the means of three separate experiments in which triplicate determinations were done.

and 1.190.05 pmol/mg protein for 150 sec, respectively. In contrast, when BBM and BPM were incubated in the presence of increasing concentrations of sucrose, the arachidonic acid incorporation decreases dramatically in both membranes in a linear relation. When the curve of this representation is drawn, the ‘y-axis’ intercept represents the binding to these membranes. Thus, the binding to BBM represents about 39 per cent of the total incorporation value and it represents about 27 per cent in BPM. The difference represents a real incorporation by both syncytiotrophoblast vesiculated membranes. DISCUSSION The bipolar syncytiotrophoblast of human placenta is the main barrier between the mother and the fetus. It constitutes the

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Table 3. Arachidonic acid incorporation in brush border and basal plasma membranes in presence of Na + and ATP (sodium and calcium salts) of human term placenta. The fatty acid was bound to albumin in a 6 : 1 molar ratio. The experiment were performed with 20 g proteins of BBM or BPM, for 150 sec at 37C. Results are expressed as the mean in which triplicate determinations of three placentae were performed Incorporation

BBM (pmol/mg)

BPM (pmol/mg)

Incorporation basal Incorporation+Na + Incorporation+ATP-Na + Incorporation+ATP-Na + +Na + Incorporation+ATP-Ca2+ Incorporation+ATP-Ca2+ +Na +

0.750.10 7.680.98a 4.750.35a,b 8.540.45a 5.200.40a,b 9.200.90a

0.480.18 6.530.20a 4.400.84a,b 7.890.15a,b — —

P<0.05 versus basal; bP<0.05 versus Na + .

Arachidonic acid incorporation (pmol/mg)

a

2.0

1.5

1.0

0.5

0

2.5

5.0 7.5 [Sucrose] (M–1)

10.0

12.5

Figure 5. Effect of increasing concentrations of sucrose (0–3.2 ) and ATP conjointly on [3H]-arachidonic acid incorporation into BBM and BPM. Membrane vesicles (20 g) were preloaded with 100 m sucrose and preincubated in the presence of external increasing concentrations of sucrose, with or without ATP, during 3 min. The experiments was performed with BBM in absence ( ) or presence () of ATP or with BPM in absence () or presence () of ATP, at 37C during 150 sec. The arachidonic acid incorporation was initiated by the addition of 20 n [3H]-arachidonic acid for 150 sec at 37C. The fatty acid was bound to albumin in a 6 : 1 molar ratio. Results express the means of three separate experiments in which triplicate determinations were done.

BBM facing the maternal circulation and BPM facing the fetal circulation. The transplacental exchange between the mother and her fetus implies obligatory transport of nutrients through these two membranes. We presume that fatty acids are selectively transported through this main selective barrier, like many other nutrients (DeGrella and Light, 1980a), lipids (Lafond et al., 1994a; Stephenson, Stammers and Hull, 1991) and ions (Lafond, Leclerc and Brunette, 1991). In 1986, Coleman (1986) suggested that the placental trophoblast could be implicated in fatty acid transport to the fetus. Although many studies on fatty acid transport have been carried out in whole perfused placenta, the originality of the present study is the direct measurement of fatty acids incorporation by vesiculated BBM and BPM prepared from human term placenta.

Thus, in this study, it is very important to discriminate between BBM and BPM, since these two membranes are selectively implicated in the transfer of nutrients from the mother to her fetus, and have some distinct functions. In this paper, we demonstrated that syncytiotrophoblast BBM and BPM incorporated arachidonic acid with the same efficiency (Table 2). Effectively, Campbell et al. (1998) have demonstrated that placental membranes preferentially bind, in respective order, the arachidonic acid, linoleic acid, -linolenic acid and oleic acid. Moreover, Haggarty et al. (1997) have confirmed these results and suggested that similar incorporation by both membranes is due to the affinity of the membrane structure for the arachidonic acid. However, some of those results contrast with another study from our laboratory showing that linoleic acid is preferentially incorporated by BBM compared with BPM (Lafond et al., 1994a). This suggests that the mechanisms involved in these fatty acid incorporations are different. Effectively, affinities of both mechanisms are different, since the Km of arachidonic acid is 20 n while that of linoleic acid is 8 . The affinity found for arachidonic acid is similar to those reported by Stremmel, Kachwa and Berk (1983) and Stremmel and Theilmann (1986) for oleic acid, which is about 80 n. Similarly, Bojesen and Bojesen (1995) have shown that the arachidonic acid incorporation in erythrocyte ghosts is different than that of palmitic acid. Indeed, the affinity of arachidonic acid for its transporting system and its binding capacity to erythrocyte ghosts are different from those of palmitic acid, and this fatty acid shows less affinity for its transporting system and a higher binding capacity than arachidonic acid (Bojesen and Bojesen, 1995). Moreover, arachidonic and linoleic acid incorporation were studied in the presence of albumin. It is known that the binding interaction between fatty acid and albumin differ for different fatty acids (Wosilait and Soler-Argilaga, 1977), thus it is possible that this binding capacity to albumin may influence the fatty acid incorporation by syncytiotrophoblast membranes. This study shows that the incorporation of arachidonic acid by both placental membranes is saturable, implying a facilitated transport mechanism. Our results are in accordance with others obtained in placenta (Campbell, Gordon and DuttaToy, 1996) and in various tissues such as hepatic cells (Stremmel and Berk, 1986; Sorrentino et al., 1989), cardiac cells (Paris et al., 1979; Sorrentino et al., 1989), adipocytes (Schwieterman et al., 1988) and endothelial cells (Groesky et al., 1994). Moreover, arachidonic acid incorporation in BBM and BPM is a rapid phenomenon, such as is seen in erythrocyte ghosts (Bojesen and Bojesen, 1995) and cardiac cells (Samuel, Paris and Ailhaud, 1976). However, the time course of arachidonic acid follows a sigmoid curve characterized by a latency phase of 20–40 sec, which may be explained by passive diffusion. Following this, a rapid increase in incorporation is observed, and a maximal value is reached after 180 sec, when more than 80 per cent of the maximal arachidonic acid incorporation is measured. This rapid increase may reflect a mechanism of facilitated transport. Two hypotheses could

Lafond et al.: ATP and Sodium Incorporation in Arachidonic Acid

explain these two phases of incorporation. Firstly, in the first 80 sec, arachidonic acid transport could occur by passive diffusion and the facilitated transport mechanism could follow, later on. Secondly, the interaction of the arachidonic acid– albumin complex with syncytiotrophoblast membranes could cause a latency phase in the facilitated transport mechanism. Moreover, to sustain this last hypothesis, some studies (Ockner et al., 1983; Weisigier, Gollan and Ockner, 1981) have implicated an albumin receptor. Thus, we can hypothesize that the binding of albumin to its receptor will induce arachidonic acid dissociation from albumin and, thereafter, the membrane fatty acid transporter will mediate the influx of arachidonic acid inside the vesiculated membranes. In order to obtain the real incorporation value, we evaluated the binding component of arachidonic acid to each bipolar membrane by an osmolarity experiment as described in Lafond et al. (1994a). The binding of arachidonic acid to both vesiculated BBM and BPM represented respectively 39 per cent and 27 per cent of the total incorporation. The proportion of this arachidonic acid binding to these membranes cannot be nonspecific binding, since the vesicles were washed with a buffer containing phloretin, which is known to have the capacity to remove the non-specific binding (Stremmel, Kwacha and Berk, 1983). The binding observed on both membranes could be related to the presence of some membrane components, such as FABPpm, CD36/fatty acid translocase (FAT) and fatty acid transport protein (FATP) (Campbell et al., 1998). In human placenta, one type of FABPpm was observed, which is only localized on the BBM (Campbell, Gordon and Dutta-Roy, 1996; Campbell et al., 1998), and its presence could be related to fetal development (Campbell et al., 1998). Moreover, it has been demonstrated that CD36/FAT and FATP are localized on both membranes (Campbell et al., 1998). Man et al. (1996) suggested that the FATP could be involved in FFA efflux and influx. Also, CD36/FAT are implicated in many biochemical processes, including the FA transport (Abumrad et al., 1993; Greenwalt, Scheck and Rhinehart, 1995). Thus, the highest binding value observed in BBM (39 per cent) could be due to the presence of FABPpm exclusively located on this membrane, while the lowest binding value observed for BPM (27 per cent) could be related to the absence of this protein. Moreover, Campbell et al. (1994, 1995a,b, 1998) showed that placental membrane FABPpm specifically binds arachidonic, oleic and linoleic acids. According to Campbell et al. (1995b), the binding of arachidonic, oleic and linoleic acids to FABPpm, in the presence of albumin, is saturable and time- and temperaturedependent. This is in accordance with our present results. Actually, there are few reports on placental membrane fatty acid incorporation and the majority of them were conducted with homogenates, perfused placenta or in vivo (DeGrella and Light, 1980b; Hull and Elphick, 1979a,b; Hull and Stammers, 1985). Thus, because we used the phloretin solution to remove the non-specific binding and we used the osmolarity experiments to evaluate the binding components, we believe that the results obtained represent a net incorporation.

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The presence of ATP is known to act positively in some transport mechanisms, while the ATP depletion causes an important decrease of oleate transport by fibroblasts (Biade et al., 1992) and bile acids incorporate by placental BBM (Marin et al., 1995). Our results with both vesiculated syncytiotrophoblast membranes revealed that ATP increases arachidonic acid incorporation by approximately sixfold. ATP is inserted in the vesiculated BBM and BPM, since membranes were vesiculated in a transport buffer containing ATP and its regenerating system, and it is also present outside the vesicles since all the ATP is not captured by the vesicles. Moreover, in accordance with the implication of sodium in the oleate transport by liver cells (Stremmel, Strohmeker and Berk, 1986), we demonstrate that the presence of Na + in the transport buffer increases the arachidonic acid incorporation in both membranes. In accordance with our results, bile acids incorporation by liver BPM was increased twofold in the presence of Na + (Blitzer and Lyons, 1985) and similar results were observed for taurine incorporation in intestine BBM (Wolffman, Hagmann and Scharrer, 1991). In the presence of ATP or sodium, the arachidonic acid incorporation by BBM and BPM was significantly increased, demonstrating a significant modification of the Vmax values, without changing the Km values. Our results are supported by the studies cited above and suggest that the increasing incorporation of arachidonic acid in BBM and BPM from human placenta is associated with an active transport of this fatty acid, and is influenced by the surrounding medium. In the experiments conducted in presence of Na + or ATP, we observed that the arachidonic acid incorporation is much more important for both membranes (BBM and BPM) as compared to the basal one. Interestingly, we observed that the increase of the arachidonic acid incorporation in BBM is not significantly different from the results obtained in the presence of Na + only, while on BPM, the increase of arachidonic acid incorporation is statistically different. This could be explained by the presence of Na + / K + ATPase. Also, the slight increase of arachidonic acid incorporation observed through BPM in the presence of both Na + and ATP suggest that the modulation of incorporation is ascribed to the presence of Na + rather than ATP. Also, to confirm a real ATP effect on the arachidonic acid incorporation, we conducted the experiments with another type of ATP, in the form of a Ca2+ salt. Any significant difference was observed in the stimulation of arachidonic acid incorporation in the presence of ATP in the form of Na + or Ca2+ salts (ATPNa+ or ATPCa2+ ). Also, the stimulatory effect of ATP combined with the Na + on the arachidonic acid was not modified, whichever form of ATP was used. In summary, our experiments demonstrated that arachidonic acid is incorporated with the same efficiency by both syncytiotrophoblastic membranes, by two different mechanisms, passive diffusion and facilitated mechanism, the latter contributing principally to this incorporation. Moreover, this incorporation is greatly increased in the presence of sodium and moderately so by ATP, showing that this mechanism is modified by the surrounding medium.

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Placenta (2000), Vol. 21

ACKNOWLEDGEMENTS The authors wish to express their gratitude to Mrs Blackburn and nursing from Service d’Obste´ trique et de Gyne´ cologie of Centre Hospitalier Universitaire de Montre´ al, pavillon St-Luc for the donation of placentae. This work was supported by grants from the National Sciences and Engineering Research Council of Canada (NSERC) and Universite´ du Que´ bec a` Montre´ al.

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