63 research outputs found
Tricyclic antidepressant and beta-adrenergic antagonist interactions with biological and model membranes
Thesis (Ph. D.)--University of Kansas, Pharmacology, Toxicology, and Therapeutics, 1984. 218 leaves : ill. ; 28 cm
Controlling drug delivery across the placenta: A commentary
A challenge in modern drug therapy is to develop strategies for safer and more selective targeting of drug delivery in pregnancy. Specifically, approaches are needed that would restrict unnecessary drug exposure to either mother or fetus. There is evidence emerging that indicates the placenta does express natural transport and metabolism processes that function to control drug and nutrient distribution between the mother and fetus. Further, in vitro techniques developed in the past ten years now provide some of the tools necessary to elucidate transport and metabolism processes typical of the human placenta. As a consequence, pharmaceutical scientists are in a position to contribute significantly to the design and development of drugs for pregnancy
MRP isoforms and BCRP mediate sulfate conjugate efflux out of BeWo cells
The breast cancer resistance protein (BCRP) and the multidrug resistance-associated proteins (MRPs) have the ability to eliminate sulfate conjugates but it is not known if this constitutes one of their roles in the placenta. To determine this, the BeWo cell line was used as a model of placental trophoblast cells and we examined the mechanisms of elimination of two common sulfotransferase substrates, 4-nitrophenol and acetaminophen. At 0.5–200 μM, neither 4-nitrophenyl sulfate nor acetaminophen sulfate affected the accumulation of the BCRP substrates BODIPY FL prazosin or mitoxantrone in BeWo monolayers, indicating a lack of interaction of BCRP with the sulfates. Efflux studies and bidirectional transport studies examining the effect of BCRP/MRP inhibitors on the efflux of intracellularly generated 4-nitrophenyl sulfate and acetaminophen sulfate, indicated that one or more of the MRP isoforms play a major role in the elimination of 4-nitrophenyl sulfate and acetaminophen sulfate across the basolateral (fetal-facing) and apical (maternal-facing) membranes respectively. BCRP played a minor role in the elimination of these two sulfate conjugates across the apical membrane. Our study shows that a yet undetermined role of trophoblast efflux transporters is the elimination of sulfate conjugates
Expression and Functional Activities of Selected Sulfotransferase Isoforms in BeWo Cells and Primary Cytotrophoblast Cells
Several cytosolic sulfotransferase enzyme isoforms are functional in placenta but there is limited information available on the utility of cultured trophoblast cells for studying sulfation. The trophoblast cell layer constitutes the rate-determining barrier for trans-placental transfer. The objective of this work was to examine the mRNA expression and enzyme activities of four sulfotransferase isoforms reported to be functional in human placenta (SULT1A1, SULT1A3, SULT1E1, and SULT2A1) in primary cytotrophoblast cells and the trophoblast-like BeWo cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine mRNA expression. Enzyme activities were assessed using the following substrates: 4-nitrophenol for SULT1A1, dopamine for SULT1A3, 17β-estradiol for SULT1E1, and dehydroepiandrosterone for SULT2A1. For 4-nitrophenol and dopamine sulfation, apparent Km values, response to inhibitors (2,6-dichloro-4-nitrophenol and sodium chloride), and thermal stability profiles indicated that 4-nitrophenol and dopamine sulfation in BeWo cells were being mediated by SULT1A1 and SULT1A3, respectively. SULT1A1 and SULT1A3 were also functional in the cytotrophoblast cells. Both at the protein and at the mRNA levels, SULT1A1 was more abundant in BeWo cells in comparison to the primary cytotrophoblast cells. SULT1E1 and SULT2A1 mRNA were not detected in the cytotrophoblasts. SULT1E1 mRNA was weakly expressed in BeWo but there was negligible functional activity. Although SULT2A1 mRNA was abundantly expressed in BeWo, Western blot and enzyme activities revealed that the protein is not expressed in BeWo cells. The results suggest that the BeWo cells and the cytotrophoblast cells can be used to examine the roles of SULT1A1 and SULT1A3 in placental metabolism
Multidrug resistance-associated protein (MRP) isoforms and the breast cancer resistance protein (BCRP) mediate sulfate conjugate efflux out of BeWo cells
The breast cancer resistance protein (BCRP) and the multidrug resistance-associated proteins (MRPs) have the ability to eliminate sulfate conjugates but it is not known if this constitutes one of their roles in the placenta. To determine this, the BeWo cell line was used as a model of placental trophoblast cells and we examined the fate of two common sulfotransferase substrates, 4-nitrophenol and acetaminophen. At 0.5–200 μM, acetaminophen sulfate did not alter the accumulation of the BCRP substrates BODIPY FL prazosin or mitoxantrone in BeWo monolayers indicating a lack of interaction of BCRP with acetaminophen sulfate. 4-nitrophenyl sulfate increased the accumulation of BODIPY FL prazosin only at 200 μM, indicating it to be a BCRP inhibitor at high concentrations. Efflux studies and bidirectional transport studies examining the effect of BCRP/MRP inhibitors on the efflux of intracellularly generated 4-nitrophenyl sulfate and acetaminophen sulfate, indicated that one or more of the MRP isoforms played a major role in the elimination of 4-nitrophenyl sulfate and acetaminophen sulfate across the basolateral (fetal-facing) and apical (maternal-facing) trophoblast membranes respectively. BCRP played only a minor role in the elimination of these two sulfate conjugates across the apical membrane. Our study shows that a yet undetermined role of trophoblast efflux transporters is the elimination of sulfate conjugate
Drug disposition and targeting: Transport across the blood-brain barrier
The original publication is available at www.springerlink.co
Effect of Bisphenol A on Drug Efflux in BeWo, a Human Trophoblast-like Cell Line
Bisphenol A (BPA) is a monomer of polycarbonate plastics that has estrogenic activities and has been shown to be a substrate for multidrug resistant efflux mechanisms, specifically, P-glycoprotein. Since the natural hormone estrogen reverses multidrug resistance in some cell types, we hypothesized that BPA might have a similar activity in trophoblasts. We have used BeWo cells as an in vitro model for human trophoblasts and calcein AM as a substrate for drug efflux mechanism to characterize BPA interactions with placental P-glycoprotein. We found that chronic exposure of BeWo cells to BPA did not alter intracellular calcein accumulation in a fashion that would be reflective of changes in P-glycoprotein expression. Immunoblots affirmed that BPA had small effects on P-glycoprotein expression. However, BeWo cells acutely exposed to BPA pretreatment were observed to have a significantly decreased calcein accumulation. Addition of cyclosporin A, a P-glycoprotein inhibitor and substrate, completely reversed BPA's effects on calcein accumulation and resulted in a net increase, relative to controls, in calcein accumulation by the BeWo cells. BPA was found not to stimulate P-gp ATPase or alter intracellular esterases mediating calcein release from calcein AM. Therefore, our results suggested that BPA stimulated drug efflux by BeWo cells probably by direct effects on P-glycoprotein
Carrier-mediated transport of valproic acid in BeWo cells, a human trophoblast cell line
The biochemical mechanisms mediating the rapid distribution of valproic acid across placenta are not precisely known. We have characterized valproic acid transport by the human trophoblast using the human choriocarcinoma cell line, BeWo. The uptake of [14C]valproic acid by BeWo cells was found to be saturable and blocked by pre-exposure to the metabolic inhibitors, sodium azide and 2,4-dinitrophenol. Valproic acid uptake by the BeWo cells was also inhibited by the protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) but not anion exhange inhibitor. Selected monocarboxylic acids inhibited the uptake of [14C]valproic acid by BeWo cells, whereas dicarboxylic acids did not alter the uptake process. Analysis of Lineweaver-Burk plots of valproic acid uptake in the presence of benzoic acid, a marker for the monocarboxylic acid transporter, revealed a competitive process for uptake. In transcellular transport experiments, the permeation of [14C]valproic acid from the apical-to-basal side of the monolayers was signficantly greater than the permeation from basal-to-apical side. Additionally, the permeation of [14C]valproic acid from apical-to-basal side was inhibited by monocarboxylic acids and not dicarboxylic acids. The results provide biochemical evidence of a proton-dependent, saturable, and asymmetric transport system, presumed to be a monocarboxylic acid transporter, for valproic acid in a human trophoblast model
Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers
The dynamics of membrane recycling were examined
in primary cultures of brain microvessel endothelial
cells (BMECs). Because the BMEC surface was dominated
by galactosylated glycoconjugates, ricin agglutinin
(RCAI) was used as a tracer to follow the
endocytosis and recycling of RCAI binding sites.
These binding sites accounted for 75 % of the iodinatable
or most externally disposed plasma membrane
proteins. Because greater than 90 % of the RCAI that
had bound to BMECs was removed by a brief, nontoxic
treatment with galactose, the amounts and
kinetics for internalization and efflux of [125I]RCAI
were measured. Both endocytosis and efflux were
energy dependent. By using pseudo-first-order kinetics,
the £j values for RCAI binding, internalization
and efflux were 5, 18 and 13-14 min, respectively. By
comparing efflux with and without galactose present,
we found that 60 % of the RCAI binding sites that had
been internalized were returned to the cell surface
and reinternalized. Quantifying the distribution of
gold-RCAI following internalization showed kinetics
consistent with that obtained using radiolabeled
RCAI. Both horseradish peroxidase (HRP) and
gold-conjugated RCAI that had bound BMEC at 4°C
became localized within more caveolae within
2.5 min of warming to 37 °C to permit endocytosis.
With time, RCAI appeared within endosomes and
tubules and vesicles of which some were located in
the trans-Golgi network (TGN). The distribution of
HRP-RCAI contrasted with that of free HRP, which
was not routed to the TGN. The absence of RCAI
conjugates in association with the basolateral membrane
domain suggested the presence of functional
tight junctions and maintenance of polarity throughout
the duration of these experiments. These results
showed that membrane recycling was more extensive
and much slower than fluid-phase endocytosis in
cultured BMECs. Moreover, we found that endocytosis
of membrane by BMECs in culture was similar
to that reported for brain endothelium in vivo in
that a fraction of the cell surface membrane was
routed to the TGN
Receptor-mediated angiotensin II transcytosis by brain mcrovessel endothelial cells
ROSE, J. M. AND K. L. AUDUS. Receptor-mediated angiotensin II transcytosis by brain microvessel endothelial cells. PEPTIDES __( ) _____, 199_. - Angiotensin II (Ang II) uptake and transport across monolayers of bovine brain microvessel endothelial cells (BMECs) was demonstrated. Ang II transport was linear up to 2 hours, saturable with a Km of 1.7 nM, and tended to be polarized with the apical-to-basolateral transport being greater. [3H]-Ang II transport was found to be inhibited by excess unlabeled Ang II, by the Ang II analog sarathrin, and by the endocytic inhibitor phenylarsine oxide. Ang II-(2-8) and -(3-8) were shown to significantly increase the transport of Ang II. These results demonstrate for the first time the receptor-mediated transcytosis of Ang II across brain microvessel endothelium
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