Low-affinity uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) in BeWo cells

Abstract

Understanding the mechanisms of transport processes in the placenta can improve the safety and efficacy of drug delivery during pregnancy. Functional studies of Organic Cation Transporters (OCTs) are usually carried out using radioactivity, and a fluorescent marker would add flexibility to experimental methods. As a published substrate for OCT1 and OCT2, the fluorescent compound 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) was chosen as a candidate for studying placental OCT function in BeWo cells. The expression of OCT1 and OCT2 was also investigated in BeWo cells, an established human choriocarcinoma trophoblastic cell line frequently used as an in vitro model of the rate-limiting barrier for maternal-fetal exchange of drugs and nutrients within the placenta. 4-Di-1-ASP was taken up into BeWo cells by a low-affinity, carrier-mediated process exhibiting a K m of 580 ± 110 M and V max of 97 ± 9 nmol/mg protein/30 min, and asymmetric transport was observed, with greater permeability in the apical to basolateral (maternal to fetal) direction. However, RT-PCR revealed no expression of OCT1 or OCT2 in either BeWo cells or primary cultured human cytotrophoblast cells, and OCT substrates such as TEA and choline did not inhibit the uptake of 4-Di-1-ASP. Although the uptake of this fluorescent compound in BeWo cells is not mediated by an OCT, the colocalization experiments with fluorescence microscopy and inhibition studies confirmed significant mitochondrial uptake of 4-Di-1-ASP. Transport of 4-Di-1-ASP into the nuclear region of BeWo cells was also observed, which is likely mediated by a nucleoside transporter.

Transport of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) in human respiratory epithelial cells

“…ASP + has been used as a nonradioactive substrate for monoamine and organic cation/carnitine transporters (15,16,18,(27)(28)(29)(30). In our hands, ASP + uptake was concentration and temperature-dependent and sensitive to extracellular pH and membrane potential in all cell lines studied.…”

“…The second aim of this paper was to use 4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide (ASP + ) as a non-radioactive OCT/N probe (15,16), and to study the mechanisms of organic cation transport across distinct lung epithelia, with particular emphasis on OCT/Nmediated drug-drug interactions. Commonly prescribed inhalation medicines such as β 2 -agonists and anti-cholinergic drugs carry a cationic charge at physiological pH, but have to cross the epithelial barrier in order to reach the β 2 -adrenoceptor localised to the airway smooth muscles.…”

In vitro placental model optimization for nanoparticle transport studies

“…[7][8][9] The trophoblast cell layer is the rate-limiting barrier for drug and nutrient exchange between mother and fetus, and the human choriocarcinoma trophoblastic BeWo cell line is an established in vitro model for placental transport studies of soluble material. 10 Although this model does not constitute a complete physiological system and the entire tissue microenvironment found in humans in vivo, it does represent the rate-limiting barrier of maternal-fetal exchange, irrespective of the transport mechanism through the cell layers. Due to interspecies differences in placental structure, it cannot be assumed that observations in animal placentae are directly translatable to humans; 11 therefore, this work has focused on a model of human origin.…”

“…23 In this work, we investigate the uptake and transport of fluorescent polystyrene NPs in BeWo b30 cells following model optimization specific to the application of NPs. The BeWo cell model has long been utilized for placental transport, 24 uptake, 13 efflux, 25 and expression studies; 10 however, we present a series of optimization procedures enabling the application of BeWo cells to transplacental NP transport experiments, thereby developing the use of this model beyond its conventional use with only soluble materials. The uptake of gold NPs was previously studied using the original (American Type Culture Collection) BeWo clone, 4 and the BeWo b30 clone has been utilized within an elaborate model to show indirect effects of cobalt-chromium NPs on fibroblast cells cultured behind a BeWo cell layer even though these particles did not traverse the BeWo cell barrier.…”

Exploring the interactions of nanoparticles with multiple models of the maternal--fetal interface

“…While perfusions with fullterm placentas cannot provide data regarding early gestation, placentas obtained following preterm deliveries may not represent normal tissue (Nanovskaya et al, 2008). The BeWo choriocarcinoma cell line is of human trophoblast origin and serves as an in vitro model of the rate-limiting barrier for maternal-fetal exchange (Rytting et al, 2007). Although the BeWo cell model lacks spontaneous syncytialization, connective tissue and endothelium present in placenta, it is a more convenient experimental model which can produce transport and metabolism data more rapidly than the perfusion model.…”