Isolation of Plasma Membrane Vesicles from Mouse Placenta at Term and Measurement of System A and System β Amino Acid Transporter Activity

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.

Uptake of ¹⁴C-MeAIB (0.165mM) into mouse placental vesicles. (A) Uptakes were measured in the presence (■) and absence (▾; K⁺ replacement) of an inwardly directed Na⁺ gradient over 60s. (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 60s. The gradients of the lines were not significantly different (F test). Data are expressed as mean±SEM. (C) Effect of neutral amino acids (20mM) 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).

Uptake of ³H-taurine (0.5μM) into mouse placental vesicles. (A) Uptakes were measured in the presence (■) and absence (▾; K⁺ replacement) of an inwardly directed Na⁺ gradient over 60s. (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 60s. 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 (500μM) 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).

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 40μg/lane other than mouse kidney lysate (25μg). An immunoreactive signal was seen in all human MVM lanes with a molecular weight of ∼86 and ∼67kDa. These bands were less intense in mouse placental vesicles. Signals were also observed in mouse kidney lysate at ∼145 and ∼47kDa. Film exposure was 30min. (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 5min.