Riboflavin uptake in microvillous and basal membrane vesicles isolated from full-term human placentas

Placenta (1994), 15, 137-146

Riboflavin Uptake in Microvillous and Basal Membrane Vesicles isolated from Full-Term Human Placentas A.J. MOE a, D. R. PLAS, K. A. POWELL & C. H. SMITH The Edward Mallinckrodt Department of Pediatrics, Department of Pathology, Children's Hospital, Washington Universi~ School of Medicine, St Louis, MO 63110, USA a To whom correspondenceshould be addressedat: Department of Pediatrics, Washington University School of Medicine, One Children's Place, St Louis, MO 63110, USA Paper accepted 18.8.1993

SUMMARY

Ribofavin uptake was characterized using membrane vesicles isolated from the apical (maternal-facing) and basal (fetal-facing) membranes of the syncytiotrophoblast from full-term human placentas. Equilibrium [SH]riboflavin uptake was insensitive to variations in incubation medium osmolar#y in contrast to [3H]alanine uptake into an osmotically sensitive space. Osmotic insensitiviO~ suggested riboflavin binding to a membrane component. The dissociation constant of riboflavin binding was similar in microvillous (Ka = 2 ~ ) and basal membrane vesicles (Ka = 1 ~ ) . Binding capacity was significantly higher in microvillous membranes (Bmax = ll.9pmol/mg protein). The relatively high affinity binding to the membrane vesicles may represent a first step in riboflavin transport.

INTRODUCTION The fetus must obtain riboflavin (an essential water soluble vitamin) from the mother by transfer across the placenta. Riboflavin's two major metabolites, flavine adenine dinucleotide (FAD) and flavin mononucleotide (FMN), function as crucial cofactors in cellular metabolic pathways. Riboflavin hypovitanaemia is known to occur among pregnant women (Baker et al, 1981) and riboflavin deficiencies during pregnancy have lead to congenital abnormalities in animal studies (Warkang, 1975). The mechanism of riboflavin transfer across the placenta is not well understood but uphill transport against a gradient has been inferred from higher circulating concentrations of riboflavin in fetal compared to maternal blood (Lust, Hagerman and Vilee, 1954). Saturable riboflavin transfer against a concentration gradient has been demonstrated using 0143-4004/94/020137 + 10 $08.00/0

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an in vitro perfusion system (Dancis, Lehanka and Levitz, 1985; Dancis et al, 1986; Dancis, Lehanka and Levitz, 1988). The transfer of radioactive riboflavin from maternal to fetal perfusate was faster than that in the reverse direction. Radioactivity was concentrated in the placenta where 43 to 59 per cent was metabolized to FAD and FMN, with FMN the major product (Dancis, Lehanka and Levitz, 1988). The radioactivity found in maternal and fetal perfusates, however, was in the form of riboflavin. Observations from this series of studies lead to the conclusion that differences in transport characteristics at the two surface membranes of the syncytiotrophoblast are responsible for the transplacental flux of riboflavin (Dancis, Lehanka and Levitz, 1988). As a step toward understanding the cellular mechanisms of riboflavin uptake, we have investigated riboflavin interaction with the microvillous and basal membrane of placental syncytiotrophoblast using isolated membrane vesicles. Uptake was characterized as to osmotic sensitivity and concentration dependence.

MATERIALS AND M E T H O D S T i s s u e source

Human placentas from uncomplicated full-term pregnancies were obtained immediately after labour and vaginal delivery or caesarean section under epidural anaesthesia without prior labour. Fresh villous tissue was excised free from basal and chorionic plates and used to begin the membrane isolation. Usually between 200 and 300 g of villous tissue remained after it was washed with Earle's balanced salt solution equilibrated with 95 per cent 02-5 per cent COz and passed through a meat grinder.

Preparation of microvillous membrane vesicles Microvillous membrane vesicles (MMV) were isolated by the method of Booth, Olaniyan and Vanderpuye (1980) as modified by Moe and Smith (1989). After grinding, the tissue was stirred vigorously in 1.5 volumes of 150raM NaCI. Tissue was removed by filtration and the filtrate was centrifuged sequentially at 800 g for 10 min, 10 000 g for 10 min, and 150000g for 25rain. The pellet was resuspended in 300mM D-mannitol, 2mM N-2hydroxyethylpiperazine-N')-2-ethanesulphonic acid (HEPES)-tris(hydroxymethyl)-aminomethane (Tris), pH 7.4 (mannitol buffer). A stock MgCI2 was added to yield a concentration of 10 mM. The suspension was homogenized 10 strokes with a glass-Teflon homogenizer and allowed to incubate 10 min before centrifugation at 2200g for 12 min. MMV were resuspended in mannitol buffer and frozen in liquid nitrogen.

Preparation of basal m e m b r a n e vesicles Basal membrane vesicles (BMV) were isolated according to the method of Kelley, Smith and King (1983) as described by Furesz, More and Smith (1991). The procedure was the same as that for the MMV to the point where the tissue was filtered. The washed tissue was sonicated and stirred in hypotonic medium. The remaining tissue was then incubated in 10mM EDTA and sonicated to remove the basal membrane from the basal laminae. Isolated BMV were purified by differential centrifugation, resuspended in mannitol buffer and frozen in liquid nitrogen.

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Moe et al: Riboflavin Uptakeby Placenta

Riboflavin uptake m e a s u r e m e n t s Riboflavin uptake was measured by a membrane filtration technique using glass fibre filters (Whatman GF/C, Maidstone, UK). The incubation medium contained in final concentrations: 100 mM NaCI, 100 mM D-mannitol, 2 mM MgCIz, and 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES)-tris(hydroxymethyl)-aminomethane (Tris), pH 7.4. Choline chloride or potassium chloride replaced sodium chloride in the same buffer for sodium-independent experiments. [3H]Riboflavin (44.6Ci/mmol) was added to a final concentration of 94 nM for each determination. Mannitol was added to the incubation buffer in experiments in which the effect of medium osmolarity on uptake was determined. Incubations were stopped by rapid filtration and washed four times with 2 ml aliquots of ice-cold 200 mM KC1. Radioactivity trapped on the filters was measured by liquid scintillation counting and uptake calculated after subtracting a filter blank. Data were normalized to membrane protein as measured by a modification (Markwell et al, 1978) of the Lowry method (Lowry et al, 1951). Papain treatment Papain (lmg/ml) was activated in 100mM cysteine and 50mM EDTA, pH6.2. This solution was diluted 100-fold with 300 mM mannitol, and 5 mM EDTA and 3 ml added per ml of membrane vesicles. After a 2 h incubation the solution was centrifuged at 150 000g for 25 min and the pellet resuspended in mannitol buffer. Control membrane vesicles received the identical treatment without papain. Mathematical analysis The Henri-Michaelis-Menten equation for one saturable component was fit to the data using the RS/1 program (Bolt et al, 1983) on the Washington University School of Medicine VAX computer. The program finds the least-squares solution by the MarquardtLevenberg method of iterations. The following equation was fit to the data: Bmax

X

[S]

S~=~+__ [/] + IS]

+Km

X

[S]

where Sb is [3H]riboflavin bound, [S] is the initial concentration of radioisotope, [I] is the concentration of unlabelled substrate, Ka is the dissociation constant, Kns is the coefficient of nonspecific binding, and Bmax is the maximum binding constant. In using this equation we assume the affinity constant is the same for substrate and 'inhibitor' (i.e. that the radiolabelled compound behaves the same as the unlabelled substrate). Structural analogs were compared to riboflavin binding in the absence of inhibitor by paired t-test.

RESULTS Time dependence of uptake Riboflavin uptake increased with time over a period of approximately 10 min (Figures 1 and 2). The presence or absence of an inwardly directed sodium gradient did not affect riboflavin uptake. Uptake by microvillous membrane vesicles was the same at 22~ and 37~ (Figure 1). Riboflavin uptake by microvillous membrane vesicles increased linearly between 2 and 8 s but the line fit to the data resulted in a significant positive y-intercept (Figure 3). Uptake was constant (maximal) between 10 and 90 min with both membrane preparations (Figures 1 and 2).

Placenta (1994), VoL 15

140 1.50

1.25

P-, 1.oo o

~ 0.75 UA v

degrees degrees Sodium, 37 degrees Choline, 37 degrees

9

< 0.50

Sodium, 22

9 9

n- 0.25

0.00

Choline, 22

[]

0

~o

N'

9'o

TIME (min)

Figure 1. Uptake of [3H]riboflavin (94 nM) by microviilous membrane vesicles in the presence of 100 mM NaCi or choline chloride. Data are means + SEM for membranes from two placentas determined in triplicate.

0.6 A c

o 0.4

% LU

0.2

it

9 9

Sodium Choline

0.0 TIME (rain)

Figure 2. Uptake of [3H]riboflavin (94 nM) by basal membrane vesicles in the presence of 100 mM NaCI or choline chloride. Data are means + SeM for membranes from two placentas except 90 min which was a single placenta.

Effect of medium osmolarity The effect of medium osmolarity on equilibrium uptake of [3H]alanine and [3H]riboflavin by microvillous membrane vesicles is illustrated in Figure 4. The linear decrease in alanine uptake with increasing medium osmolarity served as a control [Figure 4(b)]. In contrast riboflavin uptake was insensitive to changes in osmolarity [Figure 4(a)]. The osmotic insensitivity of riboflavin uptake suggests that radioactivity associated with the vesicles is not free in solution inside the vesicles and may be bound to a vesicle component. The contrast in osmotic sensitivity between riboflavin and alanine uptake was similar with basal membrane vesicles (Figure 5).

141

Moe et al: Riboflavin Uptake by Placenta 0.6

0.5

9

9

8

1'0

c ~Q.O.4

0.3 u.I 0.2 n 0.1

0.0

0

a

4

6

TIME (sec)

Figure 3. Uptake of [3H]riboflavin(94 riM) by microviliousmembrane vesiclesin the presence of 100 mM NaCl. Data are means + SEM for membranes from two placentas. Linear regression line was fit to the data with a coefficient of determination(R2) of 0.93.

1.084 0.8 ~ 0.6 0.40.2 0.0

1

2

3

4

2

3

4

125._'r i lOO7550-

1

25"

1

1,'o~molarity

Figure 4. Effect of medium osmolarity on (a) 94 nM [all]riboflavin and (b) 50 ~M [3H]alanine uptake by microvillous membrane vesicles at 90 rain. Data are means + SEM for three placentas. Linear regression line was fit to data with a coefficient of determination (R 2) of 0.96.

Concentration dependence on riboflavin uptake Equilibrium b i n d i n g of riboflavin was measured in the presence of increasing c o n c e n trations o f u n l a b e l l e d riboflavin. T h e s e experiments revealed a single high affinity riboflavin

142

Placenta (1994), VoL 15 0.8 a

0.6

9 9

~ 0.2"

0,0

I

Sodium Choline

2

3

4

2

3

4

r m

is0 100

1

11osmo~ar~y

Figure 5. Effect of medium osmolarityon (a) 94nM [3H]riboflavinand (b) 50 ~M [3H]alanineuptake by basal

membranevesiclesat 60 min. Data are means -+ SEMfor three placentas. Linearregressionlinewas fit to data with a coefficientdetermination(R2) of 0.98. binding site in both membranes (Figures 6 and 7). The apparent Ka (2.0 p~M) in microvillous membranes were similar to that in basal membrane vesicles (1.0 ~M). Total binding capacity (Bma~) of microvillous membrane vesicles (11.9 pmol/mg protein) was significantly higher than basal membrane vesicles (2.9 pmol/mg protein).

Effect of structural analogs on riboflavin binding The effect of unlabelled riboflavin and two structural analogs on riboflavin binding to MMV and BMV was investigated (Table 1). Significant inhibition of riboflavin binding was observed for all inhibitors with the exception of the microvillous vesicles in the presence of 25 ~M lumichrome. Neither structural analog inhibited to the extent that riboflavin did and lumiflavine appeared to be slightly more effective than lumichrome. Papain treatment of microvillous membrane vesicles Riboflavin binding was substantially more sensitive to papain treatment than calcium binding but less so than alkaline phosphatase (Figure 8). Papain is a proteolytic enzyme that can be used to remove membrane associated proteins from the surface of membrane vesicles. Microvillous membrane vesicles were used in these experiments because they are known to be greater than 90 per cent right-side out orientation (Kelley et al, 1979). If riboflavin binding sites are external proteins it should be possible to remove them with papain (Louvard et al, 1975). Alkaline phosphatase activity and protein removed served as controls for effectiveness of papain treatment (Figure 8). Calcium binding was determined as a control for protein removal from the inner surface of the vesicles since we know from other

Moe a al: Riboflavin Uptakeby Placenta

143

1.25

Kd = 2.0 micr0molar = 11.9 pmol/mg protein

1.00 c

o~ 0.75 o

,,, 0.50 I1.

u.. Jr" 0.25

0.00 0.01

0.10

1.00

10.00

100.00

1000.00

RF (uM)

Figure 6. Uptake of [~H]riboflavin (94nM) by microvilious membrane vesicles in the presence of increasing concentrations of unlabelled riboflavin. Data are means _ SEM for three placentas.

0.75

c (I)

~0.50 o

ILl

~:~ 0.25 ]

BmaxKd=2.90.99micromolar =

0.00 /

o.ol

o.io

] I

pmol/mg protein

1.6o

1o.oo

]

1oo.oo

1 oo6.oo

RF (uM)

Figure 7. Uptake of [3H]riboflavin (94 riM) by basal membrane vesicles in the presence of increasing concentrations of unlabelled riboflavin. Data are means + SEM for three placentas.

Table 1. Inhibition of riboflavin binding by lumichrome and lumiflavine Compound Control (0.94hi [3H]riboflavin) + 50 I,LMunlabelled RF + 25 Id,Mlumiflavine + 50 ~LMlumiflavine + 25 ~ i lumichrome + 50 I~Mlumichrome

MMV (pmoL/mg protein) 0.90 0.51 0.61 0.60 0.73 0.60

+ 0.09 _+ 0.04" _+ 0.12" + 0.15 a _+ 0.26b + 0.13 =

" < control, P < 0.05; b n.s. from control; n = three placentas.

BMV (pmol/mg protein) 0.49 0.33 0.37 0.36 0.43 0.40

_+ 0.09 _+ 0.09 a _+ 0.07 a + 0.08 = _+ 0.I0 a + 0.09 a

Placenta (1994), Vol. 15

144 80 70 60

q

RF binding

50

I---

Protein

,,z, 4o

Alkaline phosphatase

0r w o_ 30-

~[1~

Calcium binding

20 10-

IM[lllll

Figure 8. P e r c e n t loss o f microvillous m e m b r a n e c o n s t i t u e n t s following p a p a i n t r e a t m e n t . D a t a are m e a n s _+ SEM for t h r e e to five placentas.

studies in our laboratory that calcium binds inside the vesicles (Kamath et al, 1992). Papain was not effective in removing proteins internal to the vesicles as suggested by the calcium binding data.

DISCUSSION These experiments indicate that the placental syncytiotrophoblast microvillous and basal membranes possess relatively high affinity riboflavin binding sites on their surface. Several pieces of evidence support this interpretation. Riboflavin uptake is sodium independent, temperature independent, and is not linear over time (Figure 3). These observations, while not inconsistent with mediated transport, are generally more indicative of a binding process. More conclusive evidence is the insensitivity of riboflavin uptake to changes in medium osmolarity. This indicates that riboflavin is not free in solution within the vesicles but is in some way associated with a vesicle component. This uptake and binding process is saturable and of relatively high affinity. Finally the effect of papain treatment on riboflavin uptake indicates that a substantial portion of the bound riboflavin is associated with the outer surface and therefore does not cross the plasma membrane. The high affinity of the binding found in our study suggests that it participates in riboflavin transport across the placental syncytiotrophoblast. In vitro perfusion experiments (Dancis, Lehanka and Levitz, 1985; Dancis et al, 1986; Dancis, Lehanka and Levitz, 1988) as well as in vivo studies (Zempleni, Link and Kubler, 1992) demonstrate that riboflavin is transported across the placenta by a saturable but poorly understood mechanism. The kinetics of riboflavin binding to microviUous and basal membrane vesicles are consistent with the conclusion that binding may participate in transplacental flux. First, the binding capacity is significantly higher for microvillous membranes compared to basal membranes, as would be expected for transfer in the maternal to fetal direction. This difference would be magnified in vivo by the sevenfold microvillous surface enlargement factor (Teasdale and Jean-Jacques, 1985) assuming the same density per unit surface area in the two membranes. Secondly, the apparent Kd for binding to both membranes is within the normal

145

Moe et ak Riboflavin Uptakeby Placenta

range for plasma riboflavin concentrations (Baker et al, 1975), indicating that substantial binding will occur at the concentrations in maternal and fetal circulations. The effect of structural analogs on riboflavin binding is also suggestive of a process that may be linked to transport. That lumiflavine is slightly more effective as an inhibitor is consistent with transport studies (Said and Arianas, 1991; Daniel and Rehner, 1992). Binding to a membrane receptor has been proposed as a mechanism for membrane transport of other vitamins (Anderson et al, 1992). In this putative system, folate is bound to receptors on the membrane which are then internalized in caveolae. The apical membrane preparation is known to contain only the microvilli and lack most of the internal structures of the cell. Thus the translocation step in the placental transport process may require other membrane components not retained during the isolation procedure, leading us to observe the binding but not the translocation step in riboflavin transport. Our observations suggest that the placental riboflavin transport mechanism differs from those in other epithelia. A sodium-dependent riboflavin transporter has been reported for human (Said and Arianas, 1991) and rat intestine (Daniel and Rehner, 1992). A sodiumindependent electroneutral transporter has recently been found in rabbit intestine (Said, Mohammadkhani and McCloud, 1993). The kidney may reabsorb riboflavin by a cyclic organic acid transporter (Spector, 1982), an activity which has not been reported in placenta. In contrast to our observations these transporters are known to transfer riboflavin into an osmotically sensitive space. Although the concentration of riboflavin is much higher in fetal plasma than in maternal plasma the concentration of total riboflavin (including F M N and FAD) are similar in the two circulations (Zempleni, Link and Kubler, 1992). Thus, riboflavin metabolism within the placenta could also participate in transepithelial flux of this vitamin. We have described a high affinity riboflavin binding process on the microviUous and basal membrane surface of placental syncytiotrophoblast. Transport into an osmotically active space was not observed. Our findings suggest that the mechanism of transplacental flux of riboflavin is complex and requires further investigation, especially given its importance for normal fetal growth and development.

ACKNOWLEDGEMENTS We wish to acknowledgeSiddharth Kamath for his technicalhelp. This workwas supportedby HD 07562 (CS) and HD 27258 (AM) fromthe National Institute of Child Health and Human Development.

REFERENCES

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