62 M. Desforges and others J Physiol 587.1 also in a dietary model of IUGR in the rat (Jansson et al. 2006), fetal growth restriction is preceded by alterations in placental system A activity. Molecular characterization of system A has revealed that there are three highly homologous protein sub- types of this Na+-coupled neutral amino acid transporter known as SNAT1, SNAT2, and SNAT4, which are encoded by three members of the SLC38 gene family (SLC38A1, SLC38A2, and SLC38A4, respectively) (Hediger et al. 2004; Mackenzie & Erickson, 2004). SLC38A2 is expressed ubiquitously in mammalian tissues (Hatanaka et al. 2000; Sugawara et al. 2000a) and expression of SLC38A1 has been shown in various tissues including human placenta (Wang et al. 2000; Desforges et al. 2006). Functionally, SNAT1 and SNAT2 operate via similar mechanisms (Hatanaka et al. 2000; Wang et al. 2000; Yao et al. 2000). SNAT4 has a lower substrate afﬁnity for neutral amino acids and MeAIB than SNAT1 and SNAT2 (Sugawara et al. 2000b; Hatanaka et al. 2001) and also has a unique ability amongst the other system A isoforms to interact with cationic amino acids in a Na+-independent manner (Hatanaka et al. 2001). It was originally thought that SLC38A4/SNAT4 was a liver-speciﬁc isoform (Hatanaka et al. 2001). However, we have demonstrated expression in human placenta (Desforges et al. 2006), and expression in rat (Novak et al. 2006) and mouse placenta (Mizuno et al. 2002; Smith et al. 2003) has also been documented. The relative contribution of each SNAT isoform to total placental system A activity is not known; indeed it is not certain that all three isoforms are active. We have previously shown that SNAT4 protein is localized to the MVM in human placenta at ﬁrst trimester and term (Desforges et al. 2006), a location consistent with a role in uptake of amino acids into the syncytiotrophoblast. Our laboratory has also demonstrated SNAT2 protein localization to the syncytiotrophoblast of term placenta (Champion et al. 2004) but localization in ﬁrst trimester is unknown and SNAT1 localization has yet to be investigated. System A activity in human placental MVM increases between ﬁrst trimester and term (Mahendran et al. 1994) coinciding with the increased fetal nutrient demand as gestation advances and fetal growth rate accelerates. Our previous investigations have shown SNAT4 protein expression increases in placental homogenates (containing a mixture of all placental cell types) between ﬁrst trimester and term (Desforges et al. 2006) suggesting amino acid transport by this isoform could be particularly important for maintaining normal fetal growth. Furthermore, in a mouse model of IUGR which displays alterations in placental structure and function similar to those found in human IUGR (Sibley et al. 2004), altered placental MeAIB transport is speciﬁcally associated with altered Slc38a4 mRNA expression (Constancia et al. 2005). In the current study we have used immuno- histochemistry to compare localization of SNAT1, SNAT2 and SNAT4 in ﬁrst trimester and term human placenta and then examined any changes in the speciﬁc localization of SNAT4 to the MVM between these two gestations using Western blotting. We then performed experiments to test the hypothesis that the SNAT4 isoform is functional in normal human placenta and to estimate its contribution to total system A activity both in the ﬁrst trimester and at term. Deﬁnitively identifying the contribution of SNAT4 to total system A transport in placenta, which expresses all three SNAT isoforms, is complicated by their overlapping substrate speciﬁcities. Therefore our initial experiments were performed using isolated MVM vesicles which allowed optimization of experimental conditions, outside the normal physiological range, to measure SNAT4 activity. Our experimental approach was to exploit the unique property of SNAT4 to transport cationic amino acids as well as MeAIB by measuring the arginine-inhibitable component of MeAIB uptake by MVM vesicles. Subsequently, experiments were performed using intact placental villous tissue fragments to examine whether SNAT4 activity could be measured under more physiological conditions in tissue from ﬁrst trimester and term pregnancy. Methods Chemicals All chemicals were purchased from Sigma-Aldrich Co. Ltd (Poole, UK) or VWR International (Lutterworth, UK) unless otherwise stated. Tissue acquisition and ethical approval All tissue was obtained with written informed consent as approved by the Central Manchester Research Ethics Committee (REC Ref: 03/CM/031). Gestational age was estimated from the date of last menstrual period and conﬁrmed by ultrasound dating. First trimester placentas (6–13 weeks gestation) were obtained following elective medical or surgical termination of pregnancy. Term placentas (38–40 weeks gestation) were collected following caesarean section or vaginal delivery from uncomplicated singleton pregnancies. Immunohistochemistry Placental tissue samples from ﬁrst trimester and term were ﬁxed in 10% neutral buffered formalin overnight at 4◦C before thorough washing in Tris buffered saline (TBS) and parafﬁn embedding. Sections of 5 μm were de-waxed and rehydrated prior to antigen retrieval by microwaving for 2 × 5 min at 800 W in 0.01 M citrate buffer (pH 6.0). Non-speciﬁc staining was prevented by quenching C 2009 The Authors. Journal compilation C 2009 The Physiological Society