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 Km of 580 ± 110 μM 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 891-900 |
| Number of pages | 10 |
| Journal | Biochemical Pharmacology |
| Volume | 73 |
| Issue number | 6 |
| DOIs | |
| State | Published - Mar 15 2007 |
| Externally published | Yes |
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Keywords
- BeWo cells
- Drug transport
- OCT1
- OCT2
- Organic cation transporters
- Placenta
ASJC Scopus subject areas
- Pharmacology
Cite this
Low-affinity uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) in BeWo cells. / Rytting, Erik; Bryan, Jordan; Southard, Marylee; Audus, Kenneth L.
In: Biochemical Pharmacology, Vol. 73, No. 6, 15.03.2007, p. 891-900.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Low-affinity uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) in BeWo cells
AU - Rytting, Erik
AU - Bryan, Jordan
AU - Southard, Marylee
AU - Audus, Kenneth L.
PY - 2007/3/15
Y1 - 2007/3/15
N2 - 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 μM 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.
AB - 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 μM 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.
KW - BeWo cells
KW - Drug transport
KW - OCT1
KW - OCT2
KW - Organic cation transporters
KW - Placenta
UR - http://www.scopus.com/inward/record.url?scp=33846875251&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33846875251&partnerID=8YFLogxK
U2 - 10.1016/j.bcp.2006.11.020
DO - 10.1016/j.bcp.2006.11.020
M3 - Article
C2 - 17174940
AN - SCOPUS:33846875251
VL - 73
SP - 891
EP - 900
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
SN - 0006-2952
IS - 6
ER -