Isolation of plasma membrane vesicles from mouse placenta at term and measurement of system A and system beta amino acid transporter activity

Abstract

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and beta, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl(2) precipitation and centrifugation. Vesicles were enriched 11.3+/-0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system beta activity in mouse placental vesicles, measured as Na(+)-dependent uptake of (14)C-methylaminoisobutyric acid (MeAIB) and (3)H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system beta activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

Fig. 1

Alkaline phosphatase distribution in mouse placenta. (A) Intense reaction product, indicative of alkaline phosphatase activity, was localised between trophoblast layers I and II (black arrows) with deposits clearly visible between the plasma membrane infoldings of layer II. The boxed area is magnified in Fig. 1C. (B) No reaction product was observed in the absence of substrate. (C) Reaction product distribution within layer II. I, II, III indicate the three trophoblast layers; FC, fetal capillary; E, fetal capillary endothelium; MBS, maternal blood space; rbc, red blood cell.

Fig. 2

Uptake of ¹⁴C-MeAIB (0.165 mM) into mouse placental vesicles. (A) Uptakes were measured in the presence (■) and absence (▾; K⁺ replacement) of an inwardly directed Na⁺ gradient over 60 s. (B) Linearity of Na⁺-dependent ¹⁴C-MeAIB uptake into mouse placental (r² = 0.95, p < 0.05, solid line; n = 6) and human MVM vesicles (r² = 0.98, p < 0.05, dashed line; n = 6) over 60 s. The gradients of the lines were not significantly different (F test). Data are expressed as mean ± SEM. (C) Effect of neutral amino acids (20 mM) on Na⁺-dependent ¹⁴C-MeAIB uptake into mouse placental and human MVM vesicles. Data is expressed as mean + SEM with (n) given above bars. In both mouse and human vesicles uptake of ¹⁴C-MeAIB was significantly reduced by each amino acid (p < 0.01, One-way ANOVA with Dunnett's multiple comparison test).

Fig. 3

Uptake of ³H-taurine (0.5 μM) into mouse placental vesicles. (A) Uptakes were measured in the presence (■) and absence (▾; K⁺ replacement) of an inwardly directed Na⁺ gradient over 60 s. (B) Linearity of Na⁺-dependent ³H-taurine uptake into mouse placental (r² = 0.90, p < 0.05, solid line; n = 6) and human MVM vesicles (r² = 1.0, p < 0.005, dashed line; n = 5) over 60 s. The gradients of the lines were significantly different (p < 0.0005, F test). Data are expressed as mean ± SEM. (C) Effect of β-amino acids (500 μM) on Na⁺-dependent ³H-taurine uptake into mouse placental and human MVM vesicles. Data is expressed as mean + SEM with (n) given above bars. In both mouse and human vesicles uptake of ³H-taurine was significantly reduced by each amino acid (p < 0.01 One-way ANOVA with Dunnett's multiple comparisons test).

Fig. 4

Western blot comparing TAUT expression in mouse placental vesicles to human placental MVM. (A) A representative Western blot of four mouse placental vesicle isolates (M) and MVM isolates from human placenta (H) probed for TAUT, with mouse kidney lysate (K) included as positive control. Protein loading was 40 μg/lane other than mouse kidney lysate (25 μg). An immunoreactive signal was seen in all human MVM lanes with a molecular weight of ∼86 and ∼67 kDa. These bands were less intense in mouse placental vesicles. Signals were also observed in mouse kidney lysate at ∼145 and ∼47 kDa. Film exposure was 30 min. (B) Negative control showing that all TAUT signals were abolished by excess blocking peptide. (C) The same blot re-probed for β-actin showing immunoreactive signal in all samples. Film exposure was 5 min.