J Physiol 587.1 SNAT4 function in human placenta 67 SNAT4-speciﬁc MeAIB uptake by MVM vesicles The arginine-inhibitable component of system A-speciﬁc [14C]MeAIB uptake, representing SNAT4-mediated trans- port (mean ± S.E.M.), in ﬁrst trimester and term vesicles was 0.083 ± 0.014 and 0.06 ± 0.01 pmol (mg protein−1 (30 s)−1, respectively (P = 0.24, unpaired t test). Figure 3D shows that when these data are expressed as a percentage of system A-speciﬁc [14C]MeAIB uptake, the SNAT4 contribution to total system A activity was signiﬁcantly higher in ﬁrst trimester MVM vesicles compared to term vesicles under these experimental conditions. MeAIB uptake by human placental villous fragments Figure 4A shows that Na+-dependent [14C]MeAIB uptake, representing system A-mediated transport, by term placental villous fragments is signiﬁcantly higher than uptake by ﬁrst trimester fragments. This increase could not be attributed to differences in fragment size (Greenwood & Sibley, 2006) as the protein content of ﬁrst trimester and term placental villous fragments was comparable (mean ± S.E.M. = 501.8 ± 46.3 and 625.7 ± 45.6 μg, respectively, unpaired t test, P = 0.07). SNAT4-speciﬁc MeAIB uptake by villous fragments In the presence of 5 mM MeAIB, Na+-dependent [14C]MeAIB uptake by term villous fragments was not signiﬁcantly different from zero (−1.1 ± 2.5 pmol (mg protein)−1 (20 min)−1, P = 0.38, Wilcoxon’s signed rank test, n = 3) whereas Na+-dependent [14C]MeAIB uptake by ﬁrst trimester villous fragments in the presence of 5 mM MeAIB was 43.9 ± 12.7 pmol (mg protein)−1 (20 min)−1 (Fig. 4B). These data suggest that, under physiological conditions, there is a contribution of SNAT4 to system A-mediated transport in ﬁrst trimester placenta, whereas in term placenta, SNAT4 activity is minimal. SLC38A4 mRNA expression in human tissue The ampliﬁcation product generated following SLC38A4 PCR was visualized as a single band of appropriate size (152 bp) following agarose gel electrophoresis (Fig. 5A). The data reveal SLC38A4 mRNA is expressed in all human tissues examined, including human placenta and liver as positive controls (Desforges et al. 2006). The ampliﬁcation product generated following β-actin PCR was also visualized on agarose gel (Fig. 5B) and conﬁrmed cDNA integrity in all samples. Discussion We have used immunohistochemical staining to conﬁrm all three SNAT isoforms are present in the syncytiotrophoblast MVM of human placenta during ﬁrst trimester and at term suggesting each has the potential to mediate amino acid transport from maternal blood in the intervillous space, into the placenta, and towards the fetus. We proceeded to design experiments to explore whether Figure 2. SNAT4 expression in ﬁrst trimester and term MVM Western blot of 4 ﬁrst trimester (F) and 4 term (T) MVM samples probed for A, SNAT4 and B, β-actin using BeWo (B) as a negative control for SNAT4 expression. After 2 s exposure a single immunoreactive signal was observed in all lanes at the expected size of ∼45 kDa for β-actin. After 5 min exposure a single immunoreactive signal representing SNAT4 protein was observed in all MVM samples at ∼70 kDa. C and D, bar chart to show densitometric analysis of SNAT4 (C) and β-actin (D) protein expression in ﬁrst trimester and term MVM samples (median and interquartile range, n = 4 for each group). ∗P < 0.05, Mann–Whitney U-test. C 2009 The Authors. Journal compilation C 2009 The Physiological Society