70 M. Desforges and others J Physiol 587.1 isoform has a particularly important role in delivering amino acids to the fetus in early pregnancy, and secondly, that the gestational increase in system A transporter activity between ﬁrst trimester and term is likely to be mediated by up-regulation of SNAT1 and/or SNAT2. It will therefore be important to determine the contribution of these particular SNAT isoforms to system A transport across MVM during normal pregnancy in order to further understand the complex gestational changes associated with this transporter. MVM vesicles lack the intracellular regulatory components present in intact placental tissue which inﬂuence amino acid transporter activity. Therefore, in vivo, the absolute contribution of SNAT4 to total system A activity may be somewhat different from that suggested by the vesicle data (i.e. approximately 70% in ﬁrst trimester compared to 33% at term). To address this issue we performed experiments designed to measure SNAT4 activity under more physiological conditions in villous fragments from human placenta. Any measurable Na+-dependent uptake of [14C]MeAIB (0.01 mM) in the presence of 5 mM MeAIB was considered to be SNAT4-mediated system A activity based on kinetic data showing that SNAT1 and SNAT2 activity is saturable at this concentration of MeAIB (Hatanaka et al. 2000; Wang et al. 2000). It is therefore interesting that there was measurable Na+-dependent uptake of [14C]MeAIB by ﬁrst trimester villous fragments but not by term villous fragments in the presence of 5 mM MeAIB. These data suggest there is little or no SNAT4-mediated transport of neutral amino acids in term placenta and higher SNAT4-mediated transport in ﬁrst trimester placenta, a trend which agrees well with the MVM vesicle data. Expression of SNAT4 protein in term MVM does suggest a functional role in amino acid transport and therefore it is reasonable to speculate that SNAT4 could mediate the transport of cationic, rather than neutral, amino acids at term. Although this is a possibility, our laboratory has previously demonstrated evidence for only two transport systems for cationic amino acids in the MVM of term human placenta, identiﬁed to be systems y+ and y+L (Ayuk et al. 2000). Numerous studies have shown there is reduced MVM system A activity in pregnancies associated with IUGR compared to normal pregnancy (Dicke & Henderson, 1988; Mahendran et al. 1993; Glazier et al. 1997; Jansson et al. 2002). In a mouse model of IUGR which displays alterations in placental structure and function that are similar to those found in human IUGR (Sibley et al. 2004), reduced placental system A activity is speciﬁcally associated with altered Slc38a4 expression (Constancia et al. 2005). Furthermore, ablation of the major placental promoter for Slc38a4 in mice is associated with fetal growth restriction (G. Kelsey and M. Constaˆncia, personal communication). These data suggest the SNAT4 isoform of system A plays an important role in the provision of amino acids for normal fetal growth in mice. In mouse placenta Slc38a4 is imprinted, being paternally expressed (Mizuno et al. 2002; Smith et al. 2003), which further suggests a role in promoting fetal growth (Coan et al. 2005; Fowden et al. 2006). It will therefore be interesting to investigate placental SLC38A4/SNAT4 expression in human IUGR and measure SNAT4 activity across MVM using the methods described in this study, in conjunction with the fragment experiments also described, to determine any differences from normal pregnancy. One study has shown placental SLC38A1 and SLC38A2 mRNA expression in human IUGR is comparable to that in normal pregnancies but the authors did not investigate SLC38A4 expression (Malina et al. 2005). As shown in the current study, there is broad distribution of SNAT isoforms in human placenta highlighting the importance of performing functional assays in parallel to expression studies. Development of isoform-speciﬁc knockdown protocols for each of the three SNAT isoforms in cytotrophoblast cells and intact placental tissue will greatly assist in deﬁning the relative contribution of each to placental system A activity over gestation and in relation to fetal growth anomalies. SLC38A4 mRNA is expressed in various tissues from mouse (Mizuno et al. 2002) and also cattle (Zaitoun & Khatib, 2006). In the current study we demonstrated expression of SLC38A4 in various human tissues previously shown to be devoid of SLC38A4 expression (Hatanaka et al. 2001). These observations conﬁrm this isoform of system A should no longer be considered liver-speciﬁc, and suggest it may be important for the transport of amino acids required for metabolism and protein synthesis in a variety of tissues. The experimental approaches described in this study for measuring SNAT4 activity could be applied to other human tissues besides placenta. This would be useful for identifying the involvement of SNAT4 in altered system A activity associated with certain diseases. For example in diabetes there is increased system A activity in the liver (Handlogten & Kilberg, 1984; Rosenthal et al. 1985; Schenerman & Kilberg, 1986), a key gluconeogenic organ. As gluconeogenesis primarily involves the conversion of amino acids and the recycling of glucose-derived carbon skeletons to glucose (Felig, 1975), with alanine an important glucose precursor, an altered system A activity in the liver could impact on the rate of alanine-supported gluconeogenesis and inﬂuence circulating glucose levels. SLC38A2 and SLC38A4 mRNA expression is increased in experimentally induced diabetic liver (Varoqui & Erickson, 2002). It would therefore be interesting to investigate whether the increased hepatic system A activity in diabetes can be attributed to increased SNAT4 activity using similar approaches to those described in this study. C 2009 The Authors. Journal compilation C 2009 The Physiological Society