CURRENT INVESTIGATION

Dipeptide transport in brush-border membrane vesicles isolated

from normal term human placenta

Malliga E. Ganapathy, M.B., B.S., Virendra B. Mahesh, Ph.D., D.Phil., Lawrence D. Devoe, M.D., Frederick H. Leibach, Ph.D., and Vadivel Ganapathy, Ph.D.

Augusta, Georgia

We studied the transport of glycylsarcosine by the brush-border membrane vesicles isolated from normal

term human placentas. This dipeptide resisted hydrolysis by placental membrane vesicles and was

transported intact into an osmotically responsive intravesicular space. The transport process was Na• -

independent and probably occurred down a concentration gradient. Many peptides inhibited the transport

of glycylsarcosine whereas amino acids had no effect. These results demonstrate, for the first time, the

presence of a dipeptide transport system in the human placenta. (AM J OBSTET GYNECOL

1985;153:83-6.)

Key words: Dipeptide transport, Na - independence, human placenta, brush-border vesicle

Transfer of nutrients from mother to fetus across

the placenta is very important for adequate fetal growth. Placental transfer of amino acids is crucial for

both fetal functional development and growth, as dis­turbed protein biosynthesis could result in abnormal­ities in either of these processes. Transfer of amino acids across the placenta can occur in the form of free amino acids as well as small peptides. While there are ample data to support the role of transport of small peptides in the adult intestine in the overall mainte­nance of human protein nutrition,' there are no anal­ogous studies on the importance of placental transfer of small peptides in the maintenance of intrauterine nutrition. Our preliminary data strongly indicate, for the first time, that the brush-border membrane of the syncytiotrophoblast of human placenta possesses a di­peptide transport system which is distinct from the well­established amino acid transport systems.

Methods

Brush-border membrane vesicles from normal term

human placenta were prepared according to method

of Smith et al. 2 One important difference, however, is

From the Departments of Endocrinology, Obstetrics and Gynecology, and Cell and Molecular Biology, Medical College of Georgia.

This study was supported by National Institutes of Health Grant AM 28389 to F. H. L.

This is contribution No. 0887 from the Department of Cell and Mo­lecular Biology, Medical College of Georgia, Augusta, Georgia.

Reprint requests: V. Ganapathy, Ph.D., Department of Cell and Mo­lecular Biology, Medical College of Georgia, Augusta, GA 30912.

the inclusion of a Ca+· -precipitation step in our pro­cedure, which resulted in better reproducibility of the

method. We adapted this step from the methods ap­plied to brush-border membrane vesicles isolated from the intestine and kidney. Placentas from normal preg­nancies were obtained within 30 minutes of delivery. The maternal decidua was removed and the central portion between the maternal and fetal surfaces of the placenta was used for the preparation of the mem­brane. The placental tissue was cut into small pieces and collected in a I L beaker. All subsequent steps were carried out at 4° C. The tissue was washed three times in 200 ml of Hepes/Tris buffer, 10 mmol/L, pH 7.0, containing mannitol, 300 mmol/L. After washing, the tissue was placed in 300 ml of the same buffer and agitated with a magnetic stirring bar for 30 minutes. The tissue was removed with forceps, and the liquid was filtered through six layers of cotton gauze. The filtrate was centrifuged at 1000 x g for 10 minutes in order to remove blood and cellular debris. The super­

natant was centrifuged at 10,000 x g for 15 minutes.

The supernatant was collected and centrifuged again at 86,000 x g for 30 minutes. The pellets were col­

lected in 50 ml of the Hepes/Tris-mannitol buffer and homogenized in a Dounce glass homogenizer with I 0 strokes. Then 0.5 ml of calcium chloride, 1 mol/L,

was added to this homogenate and the mixture was stirred for 10 minutes and allowed to stand for an ad­ditional I 0 minutes. This was centrifuged at 3000 x g for 15 minutes. The supernatant contained brush-bor­der membranes that were collected by centrifugation

83

84 Ganapathy et al.

= ..... Q)

+> 0 ~ 25 bl)

a .......

Q) -a 20 .E

Q)

.io=

= -s.. 15 = '"" = ~ a rn a = ..... '"" .t:J ..... ~ 5 = C' ~

2 4 6

Osmolality1

Fig. I. Effect of medium osmolality on the equilibrium uptake of glycylsarcosine.

at 86,000 x g for 30 minutes. The membrane pellets

were suspended in a small volume of preloading buffer by five passages through a 25-gauge needle. The pre­loading buffer varied with each experiment and the composition of buffer is given in the respective legend to the figure or table.

Uptake of [ 1-"C]glycylsarcosine was measured by a rapid filtration technique" with the use of Metricel membrane filters (pore size, 0.45 µm) and hydrolysis of the dipeptide by the membrane preparations was determined as described earlier. 1

Results

Alkaline phosphatase and 5' -nucleotidase were used as marker enzymes to assess the purity of the brush­

border membranes. 2 Alkaline phosphatase was en­riched 19.1-fold ± 2.2-fold and 5'-nucleotidase was en­riched 18.5-fold ± 2.8-fold over the homogenate of the

washed placental tissue. Freshly prepared membrane vesicles were used throughout the study.

Glycylsarcosine is highly resistant to hydrolysis by tissue peptidases and, therefore, has been widely used

in peptide transport studies. Hydrolysis of glycylsar­cosine by placental brush-border membranes was as­sayed by a highly sensitive radiometric method. No de­tectable hydrolysis occurred during incubation periods

l:i 15 ... Q)

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0 10 a i= -

• 2

• • 4 6 8

September I. 1985 Am .J Obstet Gynecol

10 Incubation time (min)

Fig. 2. Time course of glycylsarcosine uptake. Membrane ves­icles were suspended in Hepes/Tris buffer, 10 mmol/L, pH 7.5, containing mannitol, 300 mmol/L. Cptakc was measured in Hepes/Tris buffer, 10 mmol/L, pH 7.5, containing either sodium chloride, 150 mmol/L, or potassium chloride, 150 mmol/L. The final concentration of labeled glycylsarcosine was 5 mmol/L. •-•, K • gradient. o-o, Na - gradient.

as long as 3 hours. Therefore, any uptake of glycyl­sarcosine, observed under these conditions, repre­sented uptake of the intact dipeptide.

To test whether the dipeptide was transported from the incubation medium into the intravesicular space as opposed to bound to the vesicles, we measured the uptake at 90 minutes (equilibrium uptake) while vary­ing the medium osmolality by the addition of mannitol. The uptake decreased as a linear function of the re­ciprocal of increasing medium osmolality and indicated that there was negligible binding of the dipeptide to the vesicles (Fig. 1 ). This relationship, however, did not hold when the medium osmolality was less than 200

mosm, probably because of rupture of the vesicles un­der these conditions. The results show that intact ves­icles contained an osmotically responsive intravesicular

space that disappeared with membrane disruption. The time course of glycylsarcosine uptake into these

vesicles is shown in Fig. 2. The initial rates of uptake in the presence of either an Na• gradient or a K • gra­dient were identical. With longer periods of incubation, the uptake under K • gradient conditions was greater than that measured under Na• gradient conditions.

However, the equilibrium uptake measured at 90 min-

Volume 153 Number I

utes was the same whether an Na· or a K • gradient was present. These results contrast markedly with those obtained for amino acid uptake, which is many times greater in the presence of an Na• gradient than in the presence of a K • gradient. Moreover, uptake of amino acids in the presence of an Na• gradient exhibits a transient overshoot, which is indicative of active trans­port that was absent when we studied glycylsarcosine uptake. These data show that glycylsarcosine uptake by placental brush-border membrane vesicles is Na' -independent and probably occurs down a concentra­tion gradient.

We also studied the ability of unlabeled peptides and amino acids to inhibit the uptake of labeled glycylsar­cosine. Uptake was measured at pH 5.5 because it was maximal for glycylsarcosine at this pH (data not shown). The free amino acids glycine and sarcosine minimally inhibited the uptake of glycylsarcosine while all pep­tides, except for carnosine, caused significant inhibition (Table I). These inhibition studies clearly show that glycylsarcosine uptake by placental brush-border mem­brane vesicles is a carrier-mediated process rather than a simple diffusion. If it were a simple diffusion process, it could not have been inhibited by other peptides. Since free amino acids did not inhibit glycylsarcosine uptake, it appears that the transport systems involved in the placental transfer of amino acids and dipeptides are different. We used a I-minute incubation in these in­hibition experiments. However, it should be noted that shorter incubation periods (as low as I or 2 sec­onds) would be required for more rigorous kinetic analysis.

Comment We have demonstrated, for the first time, that the

brush-border membrane of the human syncytiotro­phoblast possesses a transport system for small pep­tides. This system is distinct from those used for amino acid transport and may, in vivo, serve as a significant conduit for amino acids.

Normal human plasma contains appreciable amounts of small peptides. The peptide content of plasma sig­nificantly increases during assimilation of dietary pro­teins as many small peptides arising from protein diges­tion escape further hydrolysis and cross the intestine in intact form. 5 During pregnancy, maternal metabo­lism undergoes adaptive changes to ensure a proper nutrient supply to the fetus. Alterations in hormonal balance result in elevated levels of circulating insulin in the mother.6 The maternal response to insulin changes during pregnancy. Early in gestation, the an­abolic effects of insulin result in increased fat deposi­tion and increased glycogen and protein synthesis while later there is increased muscle protein catabolism, pos-

Dipeptide transport in brush-border membrane vesicles 85

Table I. Effect of amino acids and peptides on glycylsarcosine uptake

GlycJlsarcosine uptake

nmollmg Addition Protein o/c

None 2.98 ::+: 0.28 100 Glycine 2.44 ::+: 0.09 82 Sarcosine 2.49 ::+: 0.19 Glycylsarcosine 1.32 ::+: 0.02 44 Glycylproline 1.25 ::+: 0.09 42 Carnosine 3.33 ::+: 0.11 112 Glycylleucine 0.91 ::+: 0.03 31 [3-Alanylglycylglycine 2.00 ::+: 0.09 67 Prolylglycylglycine 2.21 ::+: 0.18 74

Membrane vesicles were preloaded with Mes/Tris buffer, 10 mmol/L, pH 5.5, containing mannitol 125 mmol/L, and potassium chloride, 80 mmol/L. Uptake was initiated by add­ing 50 µI of membrane suspension to 200 µI of Mes/Tris buffer, 10 mmol/L, pH 5.5, containing sodium chloride, 80 mmol/L, labeled glycylsarcosine, and unlabeled peptides or amino acids. The final concentration of labeled glycylsar­cosine was 5 mmol/L and the concentration of unlabeled peptides or amino acids was 100 mmol/L. Osmolality was maintained constant by varying the mannitol concentration. Incubation time was 1 minute. The data represent means of triplicates ::+:SD.

sibly due to the increased insulin resistance of maternal tissues." The increased breakdown of muscle proteins occurs at a time when the fetus gains most of its body mass and may represent an adaptive process important for assuring adequate fetal growth.

While it is generally assumed that only free amino acids are released into the circulation during intracel­lular protein catabolism, no experimental evidence ex­ists to support this assumption. Therefore, it is quite possible that small, hydrolysis-resistant peptides are generated as well and may gain access to the maternal circulation. If this, in fact, takes place, then the con­centration of small peptides in maternal plasma may increase in the last trimester of pregnancy and may become an important source of amino acids for the fetus. Furthermore, placental tissue contains peptidases and may augment the supply of amino acids available to the fetus, once small peptides are transported across the placental brush-border membrane.

The peptide transport system may also participate in the transfer of biologically active peptides of maternal origin, whether derived from dietary or endogenous proteins. These compounds, following maternal-fetal transfer, may exert a wide range of physiologic effects on the fetus, including circulatory homeostasis, neu­roregulation, and liver function. Clinical conditions such as maternal hypertension, which alter placental anatomy, morphology, and function, may also influence the peptide transport system. Considering the impor­tant role this transport system may play in fetal metab-

86 Ganapathy et al.

olism and development, it merits further study in nor­mal and pathologic gestations.

REFERENCES

I. Silk OBA, Hegarty J E, Fairclough PD. Clark \1L. Char­acterization and nutritional significance of peptide trans­port in man. Ann Nutr \letab 1982;26:'.1'.~7.

2. Smith CH. l\;elson OM, King BF, Donohue D\I, Ruzycki SM, Kellev LK. Characterization of a microvillous mem­brane prej)aration from human placental svncvtiotropho­blast: a morphologic, biochemical and physiologic study. A~tJ 011sn:rGY:\EC01. 1977;128:190.

Bound volumes available to subscribers

September I, 1985 Am J Obstet Gynccol

3. Ganapathy V, Mendicino JF, Leibach FH. Transport ol glycyl-1.-proline into intestinal and renal brush border ves­icles from rabbit. J Biol Chem 1981;256:118.

4. Ganapathv V, Burckhardt G, Leibach HI. Characteristics of glyrylsarcosine transport in rabbit intestinal brush-bor­der membrane vesicles. J Biol Chem I 984;259:8954.

5. Gardner MLG. Intestinal assimilation of intact peptides and proteins from the diet-a neglected field? Biol Rev I 984;59:289.

6. Freinkel N. Banting Lecture, 1980, Of pregnancy and progeny. Diabetes 1980;29: I 023.

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