J Physiol 587.1 SNAT4 function in human placenta 69 Figure 4. [14C] MeAIB uptake by placental villous fragments A, Na+-dependent [14C]MeAIB uptake by ﬁrst trimester (FT) and term placental villous fragments (n = 16 in each group). These data represent system A-mediated transport of [14C]MeAIB and are presented as median and interquartile range. System A activity is signiﬁcantly higher at term compared to during ﬁrst trimester (∗P < 0.05, Mann–Whitney U-test). B, Na+-dependent [14C]MeAIB uptake by ﬁrst trimester (n = 6) and term (n = 3) villous fragments in the presence of 5 mM MeAIB, representing SNAT4-mediated activity. SNAT4-mediated activity is signiﬁcantly lower at term compared to during ﬁrst trimester (∗P < 0.05, Mann–Whitney U-test). Following consideration of the substrate speciﬁcity and kinetic characteristics of SNAT4 we deﬁned SNAT4-mediated transport by MVM vesicles as arginine-inhibitable [14C]MeAIB uptake. The strategy of using the cationic amino acid arginine as a SNAT4 sub- strate is confounded by its speciﬁcity also for the amino acid transporter systems y+ and y+L which are active in MVM of ﬁrst trimester and term human placenta (Ayuk et al. 2000). However these two cationic amino acid trans- porter systems do not support MeAIB as a substrate (Fei et al. 1995; Pfeiffer et al. 1999; Broer et al. 2000) strengthening the rationale for using this competitive inhibition approach. In the current study, experimental conditions using MVM vesicles were designed to promote MeAIB transport by SNAT4: the pH and the Na+ gradient set across MVM vesicles were optimized to promote neutral amino acid transport by SNAT4 (Hatanaka et al. 2001). Furthermore, we used a relatively high concentration of [14C]MeAIB (0.4 mM) compared to pre- vious studies of system A activity in placenta (0.165 mM) in order to achieve contribution to transport across MVM by SNAT4. This was based on functional expression studies of the cloned SNAT transporters in HRPE cells, which demonstrate SNAT4 has a relatively low afﬁnity for MeAIB (K m = 6.7 mM) compared to the other SNAT Figure 5. Ampliﬁcation products for SLC38A4 (A) and β-actin (B) PCR visualized on agarose gel using a 50-bp ladder The human tissue samples include: Br, brain; St, stomach; Lu, lung; SI, small intestine; H, heart; Ki, kidney; Pl, term placenta; Li, liver; –RT, negative control (no reverse transcriptase enzyme); NTC, no template control. SLC38A4 mRNA was expressed in all tissues. C 2009 The Authors. Journal compilation C 2009 The Physiological Society isoforms (K m = 0.89 and 0.39 mM for SNAT1 and SNAT2, respectively) (Hatanaka et al. 2000, 2001; Wang et al. 2000). In agreement with previous observations (Mahendran et al. 1994), and using a modiﬁed assay system, we have shown that total system A activity in placental MVM is higher at term than during ﬁrst trimester. Consistent with these observations, we have also demonstrated increased placental system A activity between ﬁrst trimester and term using freshly isolated villous fragments. In contrast, Ericsson et al. (2005) reported no signiﬁcant difference in Na+-dependent transport of MeAIB into villous fragments between ﬁrst trimester and term. The reason for these differing observations could be related to the size of fragments used (Greenwood & Sibley, 2006) or due to the concurrency of our experiments in contrast to the Ericsson et al. (2005) study, which compared ﬁrst trimester data to term data arising from an earlier study by Jansson et al. (2003). Despite there being increased total system A activity in human placenta between ﬁrst trimester and term, our data demonstrate that SNAT4 activity in ﬁrst trimester MVM is comparable to in term MVM; however, the contribution of SNAT4 to total system A activity is lower in term MVM compared to ﬁrst trimester. Collectively these data indicate, ﬁrstly, that this broader speciﬁcity