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J Physiol
2015 Oct 15;59320:4549-59. doi: 10.1113/JP270743.
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Glutamate cycling may drive organic anion transport on the basal membrane of human placental syncytiotrophoblast.
Lofthouse EM
,
Brooks S
,
Cleal JK
,
Hanson MA
,
Poore KR
,
O'Kelly IM
,
Lewis RM
.
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The organic anion transporter OAT4 (SLC22A11) and organic anion transporting polypeptide OATP2B1 (SLCO2B1) are expressed in the basal membrane of the placental syncytiotrophoblast. These transporters mediate exchange whereby uptake of one organic anion is coupled to efflux of a counter-ion. In placenta, these exchangers mediate placental uptake of substrates for oestrogen synthesis as well as clearing waste products and xenobiotics from the fetal circulation. However, the identity of the counter-ion driving this transport in the placenta, and in other tissues, is unclear. While glutamate is not a known OAT4 or OATP2B1 substrate, we propose that its high intracellular concentration has the potential to drive accumulation of substrates from the fetal circulation. In the isolated perfused placenta, glutamate exchange was observed between the placenta and the fetal circulation. This exchange could not be explained by known glutamate exchangers. However, glutamate efflux was trans-stimulated by an OAT4 and OATP2B1 substrate (bromosulphothalein). Exchange of glutamate for bromosulphothalein was only observed when glutamate reuptake was inhibited (by addition of aspartate). To determine if OAT4 and/or OATP2B1 mediate glutamate exchange, uptake and efflux of glutamate were investigated in Xenopus laevis oocytes. Our data demonstrate that in Xenopus oocytes expressing either OAT4 or OATP2B1 efflux of intracellular [(14)C]glutamate could be stimulated by conditions including extracellular glutamate (OAT4), estrone-sulphate and bromosulphothalein (both OAT4 and OATP2B1) or pravastatin (OATP2B1). Cycling of glutamate across the placenta involving efflux via OAT4 and OATP2B1 and subsequent reuptake will drive placental uptake of organic anions from the fetal circulation.
Figure 1.
Trans‐stimulation of [14C]glutamate efflux into the fetal circulation of the isolated perfused placenta following addition of boluses of potential exchanger substrates to the fetal circulation
A, trans‐stimulation of [14C]glutamate efflux into the fetal circulation was observed in exchange for fetal glutamate, but none of the other potential exchange substrates. Data presented represent the increase in glutamate transfer as the area under the curve (AUC) above baseline release. *P = 0.002 vs. baseline, n = 3–12. B, aspartate bolus alone did not stimulate glutamate efflux but aspartate in combination with the OAT4 and OATP2B1 substrate BSP (used in combination to inhibit glutamate reuptake by EAAT transporters) stimulated observable exchange. n = 5, *P = 0.04 vs. baseline.
Figure 2.
Expression of OATs and OATPs in term human placenta and primary cytotrophoblast
A, rtPCR amplification of cDNA from term human placenta, control tissue (kidney or liver) and no template control (NTC) for the OATs. B, rtPCR amplification of cDNA from term human placenta, control tissue (kidney, brain or liver) and NTC for OATP1A2, OATP1B3, OATP3A1, OATP4C1. C, rtPCR amplification of cDNA from term human placenta and NTC for OATP2A1, OATP4A1 and OATP2B1. D, rtPCR amplification of cDNA from term human placenta, control tissue (kidney) and NTC for AGT‐1. E, rtPCR amplification of cDNA from primary term human cytotrophoblast culture and in NTC for OATP2B1, OATP2B1, OATP4A1 and OAT4. plac, placenta; kid, kidney; liv, liver; cyto, cytotrophoblast.
Figure 3.
Trans‐stimulation of glutamate efflux by extracellular exchange substrates increases in a linear manner between 2 and 8 min
In Xenopus oocytes expressing OAT4 or OATP2B1 the addition of extracellular exchange substrate (glutamate for OAT4 and BSP for OATP2B1) led to a time‐dependent increase in [14C]glutamate efflux. In contrast, there was no efflux in the absence of extracellular exchange substrate. Data are means and SEM, n = 3.
Figure 4.
Trans‐stimulation of [14C]glutamate from Xenopus oocytes expressing OAT4
Efflux of intracellular [14C]glutamate was trans‐stimulated by the extracellular exchange substrates glutamate, estrone‐sulphate (E‐3‐S) and bromosulphothalein (BSP). *P < 0.05 vs. ND91, n = 5 independent experiments. Data represent total efflux of [14C]glutamate into buffer in 5 min.
Figure 5.
Trans‐stimulation of [14C]glutamate from Xenopus oocytes expressing OATP2B1
Efflux of intracellular [14C]glutamate was trans‐stimulated by the extracellular exchange substrates 5 mmol l−1 pravastatin, 20 mmol l−1 estrone‐sulphate (E‐3‐S) and 20 mmol l−1 bromosulphothalein (BSP). *P < 0.05 vs. ND91 Buffer, n = 5 independent experiments. Data represent total efflux of [14C]glutamate into buffer in 5 min.
Figure 6.
The glutamate gradient may drive uptake of OAT4 and OATP2B1 substrates from the fetus
Once in the placenta these can be transported to the mother in the case of xenobiotics or, in the case of DHEAS, used for placental synthesis of oestrogen. Glutamate reuptake is mediated by high affinity EAAT glutamate transporters which maintain the glutamate gradient. Glutamate efflux in exchange for OAT substrates could only be observed in the perfused placenta if reuptake via EAAT was blocked with aspartate. This suggests recycling of glutamate back to the syncytiotrophoblast.
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