16 alpha-Hydroxy-dehydroepiandrosterone sulfate (16 alpha-OH-DHEAS) mainly originates from the fetus and serves as precursor for placental estriol biosynthesis. For conversion of 16 alpha-OH-DHEAS to estriol several intracellular enzymes are required. However, prior to enzymatic conversion, 16 alpha-OH-DHEAS must enter the cells by carrier mediated transport. To identify these carriers, uptake of 16 alpha-OH-DHEAS by the candidate carriers organic anion transporter OAT4, sodium-dependent organic anion transporter SOAT, Na+-taurocholate cotransporting polypeptide NTCP, and organic anion transporting polypeptide OATP2B1 was measured in stably transfected HEK293 cells by LC-MS-MS. Furthermore, the study aimed to localize SOAT in the human placenta. Stably transfected OAT4-HEK293 cells revealed a partly sodium-dependent transport for 16 alpha-OH-DHEAS with an apparent K-m of 23.1 +/- 5.1 mu M and V-max of 485.0 +/- 39.1 pmol/mg protein/min, while stably transfected SOAT- and NTCP-HEK293 cells showed uptake only under sodium conditions with K-m of 319.0 +/- 59.5 mu M and V-max of 1465.8 +/- 118.8 pmol/mg protein/min for SOAT and K-m of 51.4 +/- 9.9 mu M and V-max of 1423.3 +/- 109.6 pmol/mg protein/min for NTCP. In contrast, stably transfected OATP2B1-HEK293 cells did not transport 16 alpha-OH-DHEAS at all. Immunohistochemical studies and in situ hybridization of formalin fixed and paraffin embedded sections of human late term placenta showed expression of SOAT in syncytiotrophoblasts, predominantly at the apical membrane as well as in the vessel endothelium.In conclusion, OAT4, SOAT, and NTCP were identified as carriers for the estriol precursor 16 alpha-OH-DHEAS. At least SOAT and OAT4 seem to play a functional role for the placental estriol synthesis as both are expressed in the syncytiotrophoblast of human placenta. (C) 2014 Elsevier Ltd. All rights reserved.

Drug treatment is critical in pregnant cows due to the possibility of a maternal-to-fetal drug transfer across the placenta. Since the (syn)epitheliochorial bovine placental barrier includes an intact uterine epithelium, which in general limits drug transfer to the fetal trophoblast, the establishment of a species- and organ-specific in vitro model like the bovine caruncular epithelial cell line 1 (BCEC-1) for testing bovine placental drug transport is desirable. P-glycoprotein (P-gp or ABCB1) is an important efflux carrier that limits drug permeability across blood-tissue barriers such as the placenta and transports a wide range of structurally unrelated compounds including many drugs commonly used in veterinary medicine. The aim of the present study was to elucidate the suitability of BCEC-1 as an appropriate in vitro model for P-gp mediated drug transport in the bovine placenta. P-gp mRNA expression was detected by RT-PCR in BCEC-1 and placental tissue. Additionally, the carrier protein was localised in the apical membrane of BCEC-1 by immunofluorescence staining with the mouse monoclonal antibody C494. Drug transport in BCEC-1 was investigated by FACS analysis using the fluorescent P-gp substrate Rhodamine 123. Inhibition of Rhodamine 123 efflux by the P-gp inhibitors Verapamil and PSC833 confirmed functional expression of P-gp in BCEC-1. Furthermore, transport measurements in the transwell-system revealed a basal-to-apical net flux of the P-gp substrate digoxin at concentrations ranging from 10nM to 10 μM. This transwell digoxin flux was inhibited by Verapamil. In conclusion, P-gp is functionally expressed in BCEC-1 and mediates a basal-to-apical flux of digoxin indicating dominant apical localization of P-gp in this cell culture model. Therefore, BCEC-1 may be an appropriate in vitro model to study drug transport across the maternal epithelium as part of the epitheliochorial placental barrier of the cow.

The orphan carrier protein Slc10a4 represents a novel member of the so-called "sodium-bile acid co-transporter family," SLC10. Slc10a4 has a close phylogenetic relationship with the liver bile acid carrier Ntcp (Slc10a1), but has no transport activity for bile acids. In a previous study Slc10a4 proved to be predominantly expressed in the rat brain, where it was localized within cholinergic neurons. However, whether this cholinergic expression pattern was exclusive for Slc10a4 and whether this protein might also be expressed in the peripheral nervous system or other peripheral organs, remained unclear. Therefore, in the present study we analyzed the expression of Slc10a4 in neuronal and non-neuronal rat tissues more systematically, employing immunofluorescence co-localization studies of the vesicular acetylcholine transporter VAChT and the vesicular monoamine transporter VMAT2. The Slc10a4 protein was found to be widely expressed throughout structures of the CNS and peripheral nervous system. In addition to cholinergic neurons in the CNS, the retina, the neuromuscular junction and parasympathetic innervations, Slc10a4 was also localized in certain monoaminergic neurons and nerve fibers in the substantia nigra, the spinal cord and sympathetic innervations. Slc10a4 expression was also detected in granules of rat peritoneal and tissue mast cells using immunofluorescence and electron microscopy. Western blot and immunoprecipitation experiments with rat brain vesicle preparations revealed that the Slc10a4 protein was expressed in synaptic vesicles where it co-localized with synaptophysin, VAChT and VMAT2. This vesicular expression pattern was also shown in the rat adrenal pheochromocytoma cell line PC12 by immunofluorescence. Based on the findings of the present study we can speculate about the function of Slc10a4 as follows: (I) Slc10a4 could be a novel vesicular transporter for cholinergic and/or various monoaminergic neurotransmitters in the central and peripheral nervous system or (II) may be involved in the regulation of the synaptic vesicle sorting or exocytosis process.

The bovine placenta produces large amounts of steroids, mainly estrone (E1) and progesterone (P-4). Specific features of bovine placental steroidogenesis are I) the expression of all enzymes needed for the production of estrogens from cholesterol in the trophoblast 2) an only marginal and temporal contribution to peripheral maternal P4 levels restricted to a period between approx. days 150-240 of gestation 3) the predominance of sulfoconjugated over free El and 4) a complementary setting of steroidogenic enzymes in the two morphologically discriminable trophoblast cell types, the uninucleated trophoblast cells (UTC) and the trophoblast giant cells (TGC). In cattle so far no definite information is available on the specific biological roles of placental estrogens and P4. However, the detection of estrogen receptors and progesterone receptors in the placentomes Suggests a role primarily as local regulators of caruncular growth, differentiation and functions. Inconsistent with a function as a caruncular growth factor is the strong evidence that in cattle placental estrogens enter the maternal compartment almost completely as estrone Sulfate (EIS), which is not active at classical nuclear receptors. Oil the other hand, E IS may be converted locally to free active estrogens via the action of steroid sulfatase (StS), which has been detected in specific parts of the bovine carancular epithelium. Alternatively or in addition, StS expression in the caruncular epithelium may serve the utilization of sulfated neutral steroid precursors (e.g. pregrienolone sulfate or cholesterol sulfate) supplied with maternal blood, thus providing free substrates for further metabolization in the adjacent trophoblast. The down-regulation of P450scc and P450c17 and the Up-regulation of 3B-HSD and aromatase during the differentiation of TGC from UTC in parallel with the up-regulation of ERE and estrogen sulfotransferase in maturing TGC suggests a function of placental estrogens primarily as auto-or intracrine regulators during this process and assigns to conjugated placental estrogens a role as inactivated by-products of TGC differentiation intended for excretion. Collectively, despite some evidence from recent studies for putative roles of placental steroids in cattle their exact functions in the bovine species remain still undefined.