64 M. Desforges and others J Physiol 587.1 1993; Kuruvilla et al. 1994; Glazier et al. 1997). Due to the different experimental conditions used in the current study, [14C]MeAIB uptakes over a 1 min time course were performed to ensure measurement at initial rate had been achieved (see Results). using a commercial kit (Bio-Rad Laboratories Ltd, Hemel Hampstead, UK). Na+-dependent [14C]MeAIB uptake by ﬁrst trimester and term fragments is expressed per mg fragment protein over 20 min, a time previously conﬁrmed to be at initial rate (Greenwood & Sibley, 2006). Measurement of SNAT4-speciﬁc activity in MVM vesicles In conjunction with the system A activity experiments described above, [14C]MeAIB uptake by MVM vesicles was also performed in the presence of 10–30 mM arginine. The arginine-inhibitable component of system A-speciﬁc [14C]MeAIB uptake by MVM vesicles was taken as a measure of SNAT4-mediated activity as this is the only system A isoform which supports this cationic amino acid as a substrate (Hatanaka et al. 2001). SNAT4 has a relatively low afﬁnity for MeAIB compared to the other SNAT isoforms and therefore [14C]MeAIB was used at a higher concentration (0.4 mM) than in previous publications (0.165 mM; Mahendran et al. 1994; Glazier et al. 1997) to ensure contribution to uptake by SNAT4. These experiments, performed under non-physiological conditions (i.e. pH 8.5, manipulated transmembrane Na+ gradient, and the use of supraphysiological concentrations of arginine), were designed to conﬁrm SNAT4 activity in MVM and provide an estimate of its contribution to system A transport. Measurement of total system A activity in placental villous fragments Villous fragments represent a more physiologically relevant model than isolated MVM vesicles because tissue architecture, cell-to-cell interactions, and intracellular signalling pathways remain intact, important for the maintenance of the normal driving forces for amino acid transport. Therefore, system A activity measurements were also performed using this model. Placental villous fragments were dissected from ﬁrst trimester and term placenta and rinsed in 1 : 3 mix of Dulbecco modiﬁed Eagle medium (DMEM)/control Tyrode’s buffer (135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 5.6 mM glucose, pH 7.4). Uptake of [14C]MeAIB (10 μm) into villous fragments over 20 min was carried out in control and Na+-free Tyrode’s buffer (135 mM choline chloride replaced NaCl, pH 7.4) as pre- viously described (Jansson et al. 2003; Ericsson et al. 2005; Greenwood & Sibley, 2006). The Na+-dependent component of [14C]MeAIB uptake, representing system A-speciﬁc uptake, was calculated by subtracting [14C]MeAIB uptake in the absence of Na+ from uptake in the presence of Na+. Fragment protein content (μg) was determined using the method of Bradford (1976) Measurement of SNAT4-speciﬁc activity in placental villous fragments [14C]MeAIB uptake by ﬁrst trimester and term fragments, in both control and Na+-free conditions, was also measured in the presence of 5 mM MeAIB. The Na+-dependent component of uptake in each condition was calculated and expressed per mg protein over 20 min. The transport function of SNAT1 and SNAT2 in human retinal pigment endothelial (HRPE) cells is saturable with increasing concentrations of MeAIB over the concentration range of 0.05–5 mM (Hatanaka et al. 2000; Wang et al. 2000) whereas the K m of SNAT4 for MeAIB is 6.7 mM. Therefore, any measurable Na+-dependent [14C]MeAIB uptake by placental villous fragments in the presence of 5 mM MeAIB was taken to be that speciﬁcally mediated by SNAT4. These experiments, performed at physiological pH in intact placental tissue, were designed to provide an estimate of placental SNAT4 activity in vivo. RT-PCR In order to examine SLC38A4 mRNA expression in other human tissues besides placenta we used RT-PCR. RNA (100 ng) from various human tissues including brain, stomach, lung, small intestine, heart, kidney, liver (Ambion, Cambridge, UK) and placenta (Stratagene), were reverse transcribed simultaneously to generate cDNA, ensuring comparable reverse transcription efﬁciency, and PCR was performed using SLC38A4-speciﬁc primers (Desforges et al. 2006). The thermal cycling conditions included an initial denaturation step at 95◦C for 10 min, followed by 40 cycles of denaturation, annealing and ampliﬁcation (95◦C for 30 s, 58◦C for 1 min, 72◦C for 30 s). β-Actin mRNA expression was also examined by PCR in the same samples as previously described (Lacey et al. 2005) to conﬁrm cDNA integrity. SLC38A4 and β-actin ampliﬁcation products were visualized by ethidium bromide staining following agarose gel electrophoresis. Statistical analysis Following Western blotting, the mean intensity of signal for immunoreactive species was assessed using Image J software. These data are presented as median and interquartile range and any difference between expression C 2009 The Authors. Journal compilation C 2009 The Physiological Society