J Physiol 587.1 SNAT4 function in human placenta 65 in ﬁrst trimester and term MVM was determined by a Mann–Whitney U -test. Raw data of [14C]MeAIB uptake by MVM vesicles over the 1 min time course are pre- sented as mean ± S.E.M. Two-way ANOVA was used to statistically analyse [14C]MeAIB uptake by MVM vesicles in the presence of arginine over the 1 min time course. The calculated data of system A-speciﬁc and SNAT4-mediated [14C]MeAIB uptake by MVM vesicles and villous fragments are presented as median and interquartile range. Any difference in system A-speciﬁc or SNAT4-mediated [14C]MeAIB uptake between ﬁrst trimester and term placenta was determined by a Mann–Whitney U -test. All statistical analyses were made using GraphPad Prism software and in all cases, P < 0.05 was considered signiﬁcant. Results SNAT1, SNAT2 and SNAT4 localization in human placenta Figure 1 shows representative images from immuno- histochemical analysis of SNAT1 (Fig. 1A–F), SNAT2 (Fig. 1G–L), and SNAT4 (Fig. 1M –R) localization in ﬁrst trimester (n = 7) and term (n = 6) placental tissue. Immunostaining was predominantly localized to the syncytiotrophoblast for all SNAT proteins. Cytoplasmic staining was observed in all samples with heterogeneous but intense MVM staining apparent for all three iso- forms (Fig. 1A, G and M ). In ﬁrst trimester placenta, the intensity of staining for SNAT1 was less in cyto- trophoblasts than syncytiotrophoblast (Fig. 1A and B) but the two cell types had similar levels of staining for SNAT2 (Fig. 1G and H ) and SNAT4 (Fig. 1O). SNAT1 staining was also detected in stromal Hofbauer cells and the vascular endothelium of ﬁrst trimester placenta (Fig. 1B and C), whilst at term, this isoform of system A was exclusively localized to syncytiotrophoblast (Fig. 1D and E). SNAT 2 and 4 were also present in endothelial cells in ﬁrst trimester placenta (Fig. 1H and N ), with faint staining persisting in larger intermediate and stem villous vessels at term (Fig. 1L and Q). Subpopulations of stromal cells were positive for SNAT2 and 4 at both stages of gestation (Fig. 1H , O and Q). Spatial immunostaining patterns were consistent in all samples examined, but considerable variation was detected in staining intensity. Negative control sections lacked detectable stain (Fig. 1F, I and R). SNAT4 protein expression in ﬁrst trimester and term MVM SNAT4 protein expression was compared in ﬁrst trimester and term MVM samples (n = 4 for each). Figure 2A shows that a band representing protein with a molecular weight C 2009 The Authors. Journal compilation C 2009 The Physiological Society corresponding to that of SNAT4 (∼70 kDa) was present in all MVM samples. There was no detectable immuno- reactive signal in the BeWo sample used as a negative control, conﬁrming antibody speciﬁcity. Densitometric analysis of the blots shown in Fig. 2A revealed that SNAT4 protein expression was higher in ﬁrst trimester MVM samples compared to term MVM (Fig. 2C), which is also visually apparent. To ensure this was not attributable to unequal protein loading, following ECL the nitrocellulose membrane was reprobed for β-actin (Fig. 2B). Densitometric analysis revealed there was no signiﬁcant difference in β-actin protein expression in ﬁrst trimester compared to term MVM samples (Fig. 2D), conﬁrming equal protein loading. System A activity in ﬁrst trimester and term MVM vesicles Total [14C]MeAIB uptake by ﬁrst trimester and term MVM vesicles was linear (P < 0.0001 for both, linear regression) over 1 min (see control uptake in Fig. 3A and B) indicating this measurement approximated to initial rate. Figure 3C shows system A-speciﬁc [14C]MeAIB uptake (calculated by subtracting uptake in the presence of 30 mM MeAIB from control uptake) by term MVM vesicles was signiﬁcantly higher than uptake by ﬁrst trimester vesicles at 30 s. Arginine-inhibitable MeAIB uptake by MVM vesicles [14C]MeAIB uptake by term MVM was inhibited by 20 mM and 30 mM arginine (Fig. 3B). Thirty millimolar arginine had the most pronounced effect and, at this concentration, also signiﬁcantly inhibited [14C]MeAIB uptake by ﬁrst trimester MVM (Fig. 3A). In experiments using term MVM vesicles, the effect of 40 mM and 50 mM arginine were also investigated but these did not cause any further inhibition of [14C]MeAIB uptake (data not shown). Inhibition of [14C]MeAIB uptake by 30 mM arginine was therefore used to calculate SNAT4-speciﬁc activity in MVM vesicles. In order to ensure that the inhibitory effect of 30 mM arginine was speciﬁc for MeAIB uptake, and not due to a general effect on amino acid uptake by MVM vesicles, we performed additional experiments to investigate the effect of 30 mM arginine on [3H]taurine uptake. Taurine is a speciﬁc substrate for the Na+-dependent amino acid transporter system β which is present in human placental MVM (Roos et al. 2004). Thirty millimolar arginine did not inhibit [3H]taurine uptake by MVM vesicles (data not shown) conﬁrming the inhibitory effect of 30 mM arginine was speciﬁc to [14C]MeAIB uptake.