Low-affinity uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) in BeWo cells.
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 Km of 580+/-110 microM and Vmax 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.
Rytting
Erik
E
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047, United States.
Bryan
Jordan
J
Southard
Marylee
M
Audus
Kenneth L
KL
D001692:Biological Transport; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D008564:Membrane Potentials; D008744:Methylamines / Q000493:pharmacokinetics*; D050811:Octamer Transcription Factor-1 / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D027701:Organic Cation Transport Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D000075102:Organic Cation Transporter 2; D011726:Pyridinium Compounds / Q000493:pharmacokinetics*; D012333:RNA, Messenger / Q000032:analysis; D014327:Trophoblasts / Q000378:metabolism*
C517027:4-(4-(dimethylamino)styryl)-1-methylpyridinium iodide; D008744:Methylamines; D050811:Octamer Transcription Factor-1; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; C494165:POU2F1 protein, human; D011726:Pyridinium Compounds; D012333:RNA, Messenger; C491013:SLC22A2 protein, human