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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Departments of Pharmacology and Toxicology (P.P., F.S., Z.F., A.L., M.N.), Analytical Chemistry (H.S.), and Biomedical Sciences (M.K., V.S.), Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Hradec Králové, Czech Republic; and Institute of Experimental Biopharmaceutics, Joint Research Center of Academy of Sciences of the Czech Republic and PRO.MED.CS Praha a.s. (Mi.N.), Hradec Králové, Czech Republic
Received December 21, 2002; accepted February 25, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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;
Ambudkar et al., 1999). In this
way, P-gp confers a multidrug resistance phenomenon (MDR) to tumor cells
against a large spectrum of anticancer drugs. Substrates transported by P-gp
include a variety of structurally and pharmacologically unrelated, hydrophobic
compounds, such as various anticancer drugs, cardiac glycosides,
-blockers, calcium channel blockers, etc.
(Ambudkar et al., 1999). P-gp
has also been detected on the membranes of a wide range of normal tissues,
such as the capillaries forming the blood-brain and blood-testis barriers,
apical surface of the gastrointestinal tract, luminal membrane of the renal
proximal tubular cells, and hepatocytes. At these sites, P-gp was found to
serve as a barrier for the entry of lipophilic xenobiotics into the body
and/or to the tissues that are sensitive to their toxic injury.
However, function of P-gp in the placental barrier has been less examined
and is still not fully understood. High levels of P-gp have been detected in
human and murine syncytiotrophoblast layers, which are the crucial parts of
the hemochorial placental barrier
(Cordon-Cardo et al., 1990;
Sugawara, 1990;
Bremer et al., 1992;
Trezise et al., 1992;
MacFarland et al., 1994;
Nakamura et al., 1997;
Lankas et al., 1998;
Myloma et al., 1999;
Smit et al., 1999;
Ushigome et al., 2000;
Tanabe et al., 2001). P-gp has
been demonstrated to be integrated in the microvillous membrane of the human
syncytiotrophoblast that faces directly maternal blood
(MacFarland et al., 1994;
Ushigome et al., 2000).
Transport activity of P-gp in the placental barrier has been examined both in
vivo and in vitro (Audus,
1999). Lankas and coworkers revealed that fetuses of CF-1 mice
lacking the mdr1a gene isoform of P-gp were susceptible to cleft
palate malformation induced by avermectin B1a. Conversely, the fetuses of
wild-type mice were completely protected against the above-mentioned teratogen
(Lankas et al., 1998).
Similarly, administration of other P-gp substrates (digoxin, saquinavir, or
paclitaxel) to mdr1a-/-/1b-/- knockout
mice revealed 2.4-, 7-, and 16-fold higher transplacental transport of these
drugs into the fetus compared with wild-type mice
(Smit et al., 1999).
The in vitro action of the placental P-gp has been demonstrated in uptake
studies using the BeWo cell line (a human choriocarcinoma trophoblastic cell
line), primary cultures of the human cytotrophoblasts
(Utoguchi et al., 2000), and
human trophoblast membrane vesicles
(Nakamura et al., 1997).
Ushigome et al. (2000) studied
the function of P-gp in the placenta using the BeWo cell line cultured in a
confluent epithelial monolayer. The study suggests that due to the one-way
functional activity of P-gp located in the apical membrane, transport of
selected P-gp substrates is higher in the basolateral-to-apical and restricted
in the apical-to-basolateral direction. On the basis of all these findings, it
is believed that P-gp expressed in trophoblast cells of the placenta limits
the entry of its substrates into the fetus by reverse pumping of the compounds
from the trophoblast layers back into the maternal bloodstream. Moreover, as
the in vitro study using BeWo cells suggests, P-gp could accelerate
transplacental passage of P-gp substrates in the feto-maternal direction. Our
previous in situ experiments demonstrated that the passage of CsA across the
rat placenta is restricted in the materno-fetal direction due to the P-gp
activity (Pavek et al.,
2001).
The aim of the present article was to examine 1) the functional activity of
P-gp in both the materno-fetal and fetomaternal directions using the dually
perfused rat placenta method, and 2) to confirm at the level of the intact rat
placental barrier the asymmetry of transplacental passage of P-gp substrates
described in the in vitro epithelial model of BeWo cells. A fluorescent dye
rhodamine 123 (Rho123), which is a well established model substrate for
testing the functional transport activity of P-glycoprotein, was used
(Masereeuw et al., 1997;
van der Sandt et al., 2000).
CsA, PSC 833 (a nonimmunosuppressive derivate of CsA), and chlorpromazine were
used as selective inhibitors/modulators of P-gp, quinidine was used as a
nonselective inhibitor for both P-gp and organic cation transporters (rOCT),
and sodium azide was used as an inhibitor of ATP mitochondrial synthesis.
Because there was only limited information on P-gp expression in the rat
placenta in the current literature, we carried out immunochemical,
immunohistochemical, and RT-PCR studies to confirm expression and localization
of P-gp gene products in the rat-term placentas.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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sek (IVAX Ltd., Opava, Czech
Republic). Quinidine sulfate (QND) and diamond fuchsin were purchased from
Lachema Ltd. (Brno, Czech Republic). Chlorpromazine chloride (CPZ), bovine
serum albumin, RPMI 1640 medium, and other substances were purchased from
Sigma-Aldrich. Tris, glycine, leupeptin, pepstatin, and phenylmethylsulfonyl
fluoride were obtained from Serva GmbH (Heidelberg, Germany). 4-Nitrophenyl
phosphate disodium salt hexahydrate was purchased from Fluka. Acetonitrile and
methanol (both HPLC grade) were purchase from Merck (Darmstadt, Germany). Stock solutions of CsA and PSC833 were used in a concentration of 1 mg/ml containing 19% of ethanol and 1% of cremorphor (v/v). The final concentration of cremorphor in perfusion media was less that 0.01%. Aqueous stock solutions of QND, CPZ, and sodium azide were used in a concentration of 10, 25, and 100 mg/ml, respectively.
Animals. All experiments were approved by the Ethical Committee of the Faculty of Pharmacy (Hradec Králové, Charles University in Prague) and were carried our in accordance with the Guide for the Care and Use of Laboratory Animals, 1996; and the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes, Strasbourg, 1986.
Pregnant Wistar rats were purchased from Biotest Ltd. (Konárovice, Czech Republic) and were bred in 12/12-h day/night standard conditions with water and pellets ad libitum. Experiments were performed on day 22 of gestation. Fasted rats were anesthetized with pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL) in a dose of 40 mg/kg administered intravenously into the tail vein.
Perfusion Method. The method of dually perfused rat placenta was
used as described previously (Mohammed et
al., 1993; Pavek et al.,
2001). Briefly, the placenta was excised and allowed to dive in
the heated Ringer saline. A catheter was inserted into the uterine artery
proximal to the blood vessel supplying the selected placenta and connected
with the peristaltic pump bringing Krebs' perfusion liquid containing 1%
bovine serum albumin from the maternal reservoir. The uterine vein, including
the anastomoses to other fetuses, was ligated behind the perfused placenta and
carefully cut so that maternal solution could leave the perfused placenta. The
chosen fetus was separated from the neighboring ones by ligatures. The
umbilical artery was catheterized using the 24-gauge catheter and connected
with the tubing by which the fetal perfusion liquid from the fetal reservoir
was supplied. The umbilical vein was catheterized in a similar manner and the
selected fetus was removed. After a successful umbilical catheterization, the
fetal vein effluent was collected into preweighted glass vials to check a
possible leakage of perfusion solutions from the placenta. In the case of
leakage, the experiment was discarded. Perfusion experiments did not last
longer than 36 min, because the integrity of the placental barrier could be
affected in later intervals. This notion is based on our previous experiments
with L-[3H]glucose, a marker of paracellular diffusion,
where the transplacental passage of L-[3H]glucose
started to significantly increase in later intervals of the perfusion
(Pavek et al., 2001). Wet
weights of the placentas used in experiments were 0.54 ± 0.13 g.
To study the influence of inhibitors on both the materno-fetal and feto-maternal transport of Rho123, experiments were carried out under both steady-state and nonsteady-state conditions as described below.
Evaluation of Rho123 Metabolism in the Rat Placenta. In our pilot experiments, we studied possible placental metabolism of Rho123 during its transplacental passage. In the experiments carried out under both steady-state and nonsteady-state conditions (for experimental designs of steady-state and nonsteady-state experiments see below), Rho123 was present in the maternal perfusion solution in concentrations of 0.65 or 1.3 µM. Fetal effluent samples were analyzed using HPLC.
Effect of CsA on Transplacental Passage of Rho123 under Nonsteady-State Conditions. After successful establishment of the dual perfusion with the Rho123-free and inhibitor-free perfusion solution, the maternal and/or fetal perfusion inflows were switched to another prewarmed perfusion reservoirs containing examined compounds. The experiments started after 15 s of delay (time 0) to make it possible to fill tubing with the new liquids. The fetal umbilical vein outflow was collected in 5-min intervals into preweighted glass vials for 25 min of the experiment.
To examine possible influence of CsA on the transplacental passage of Rho123 in the materno-fetal direction, CsA was added in a concentration of 40 µM to the maternal reservoir together with Rho123 (1.3 µM). Samples were collected in 5-min intervals from the fetal umbilical vein outflow.
The feto-maternal transplacental passage of Rho123 was followed in experiments where Rho123 was added in a concentration of 1.3 µM into the fetal solution (control experiments). The effect of CsA on the Rho123 feto-maternal passage was examined by simultaneous addition of CsA (40 µM) into the maternal solution and Rho123 (1.3 µM) into the fetal solution at time 0 of the experiment. Samples were collected in 5-min intervals from the fetal umbilical vein outflow.
Effect of CsA, PSC833, QND, CPZ, and Sodium Azide on Transplacental Passage of Rho123 under Steady-State Conditions. In experiments examining the materno-fetal passage of Rho123 under the steady-state conditions, Rho123 (0.65 µM) was brought to the perfused placenta via the catheterized uterine artery immediately after the catheterization. Sample collection started (time 0) after 15-min delay. Within this delay, steady-state conditions for transplacental passage of Rho123 were achieved as suggested by a nearly constant time course of transplacental clearances during the experiment (Fig. 3). Samples were collected in 3-min intervals from the fetal umbilical vein for 36 min of the experiment. In the 12th min of the experiment, examined P-gp inhibitors were added to the maternal reservoir to reach the concentrations of 10 µM (CsA and PSC833), 40 µM (QND and CPZ), and 5 mM (sodium azide), respectively. This experimental design enables observation of the direct effect of the selected inhibitor on the steady-state transplacental passage of Rho123 within one experiment. In addition to this, the steady-state experimental approach eliminates the variability between two groups of experiments carried out under nonsteadystate experiments. On the other hand, the effect of inhibitors on the passage of Rho123 is less evident under steady-state in comparison with the data obtained in the nonsteady-state experiments.
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For the examination of the feto-maternal passage of Rho123 under steady-state conditions, fetal solution containing Rho123 (0.65 µM) was used to perfuse the selected placenta via the catheter in the fetal umbilical artery immediately after catheterization. Sample collection started 10 min after the installation of the catheter (time 0). In the 12th min of the experiment, one of the P-glycoprotein inhibitors was added to the maternal reservoir to reach a concentration of 10 µM (CsA and PSC833), 40 µM (QND), and 5 mM (sodium azide), respectively. Samples were collected in 3-min intervals from the fetal umbilical vein for 36 min of experiments.
Our previous results suggested that inhibition of P-gp was more significant
when an inhibitor of P-gp was present in the maternal compartment rather than
in the fetal one (Pavek et al.,
2001). That is why the inhibitors/modulators of P-gp were given
into the maternal solution even in the feto-maternal experiments.
In our previous study (Pavek et al.,
2001), we also demonstrated that sodium azide (5 mM) increased the
transplacental paracellular passage of L-[3H]glucose in
later intervals, which could suggest impairment of the placental barrier.
Therefore, we conducted experiments with sodium azide only for 27 min after
catheterization.
Effect of Maternal Inflow Concentration on the Transplacental Clearance of Rho123. The dependence of the materno-fetal transplacental passage of Rho123 on the concentration of Rho123 in the maternal reservoir was examined in the steady state. Rho123 was brought to the perfused placenta via the uterine artery in various concentrations of 0.42, 0.65, 1.3, 2.0, and 4.0 µM, respectively. Samples were collected in 3-min intervals from the fetal umbilical vein for 30 min of experiments. The mean materno-fetal clearance of Rho123 was calculated for every concentration from all measured intervals.
Calculations. To describe the transfer of Rho123 across the dually
perfused rat placenta in the materno-fetal direction in both nonsteady-state
and steady-state experiments, its materno-fetal transplacental clearance
(CLmf) was calculated according to eq. 1
(Mohammed et al., 1993).
 | (1) |
 | (2) |
In the nonsteady-state experiment, the total cumulative amount of Rho123 that passed across the placenta within 25 min of the experiment was calculated to assess the influence of CsA on the transplacental passage of Rho123 in both directions. To eliminate variations of the cumulative amount caused by the weight of perfused placentas, the amount of Rho123 was expressed as the amount per wet weight of the perfused placenta (nanomoles per gram).
In the steady-state experiments, the effects of P-gp inhibitors given to
the maternal solution were evaluated from the following inhibitory ratio (eq.
3).
 | (3) |
If sodium azide was examined as an inhibitor of P-gp, both the materno-fetal and the feto-maternal experiments were performed up to 27 min, and the ratio was calculated as X12–27/X0–12.
In the studies where the effect of maternal inflow concentration on the
transplacental clearance of Rho123 was examined, the data were fitted by eq.
4.
 | (4) |
HPLC Analysis of Rho123 and Its Metabolites in the Perfusion Media.
Solid phase extraction of analytes from perfusate samples was performed
according to the method of Sweatman et al.
(1990) with slight
modifications. Visiprep solid phase extraction vacuum manifold (12-port;
Supelco, Bellefonte, PA) with SPE columns (Supelclean LC-18, 1-ml tubes;
Supelco) were used for the solid phase extraction. The dry extract in the
glass tube was reconstituted in 600 µl of the mobile phase, centrifuged,
and transferred into a vial of the autosampler. The sample (100 µl) was
injected into the chromatographic column.
Chromatographic analyses were performed using chromatograph (Thermo Separation Products, Minneapolis, MN; formerly Spectra Physics). The chromatographic system consisted of a SCM400 solvent degasser, P4000 quaternary gradient pump, AS 3500 autosampler with 100-µl sample loop, SpectraFOCUS high-speed scanning UV-visible detector, SN4000 system controller, and data station (Intel-Pentium 166 MMX, RAM 64 MB, HDD 2GB) with the analytical software ChromQuest 2.1 (ThermoQuest, Inc., San Jose, CA). A LiChroCART 125-4 mm analytical column packed with Purospher RP-18e, 5 µm and precolumn LiChroCART 4-4 mm with the same stationary phase (Merck) were used for analyses. The mobile phase was composed of acetonitrile/0.01 M phosphate buffer, pH 3 (3:7, v/v). Flow rate was 1 ml · min-1.
UV-visible detection was performed either in dual wavelength mode at 500 nm (for rhodamines) and 550 nm (for diamond fuchsin used as an internal standard) or in high-speed scanning mode (range 195–750 nm with 1-nm distance, used for UV-visible spectra collection).
The retention times of Rho110, Rho123, and Diamond fuchsin under the above-mentioned chromatographic conditions were 2.40, 4.09, and 8.44 min, respectively. The whole HPLC analysis lasted 14 min.
Fluorimetric Determination of Rho123 in Perfusion Media. A
commercial SIA system with an eight-port selection valve and a fluorometric
detector equipped with a flow cell was used for fluorometric determination of
Rho123 in samples (Sklenarova et al.,
2002).
Cell Line Cultivation and Isolation of the Membrane Fraction from the
Cells. The lymphoid macrophage cell line P388 and its resistant P-gp
expressing subline P388-MDR were gifts from Dr. St'astny (Institute of
Microbiology, Academy of Sciences of the Czech Republic). The membrane
fractions of the cell lines were used as positive and negative controls for
immunochemical determination of P-gp in the rat placenta. Cells were cultured
as was reported previously (St'astny et
al., 1999).
Isolation of the Total Membrane Fraction from the Rat Placentas. Rat placentas were collected on the 22nd day of gestation and were homogenized in ice-cold buffer (1:1, v/w) containing 250 mM sucrose, 10 mM Tris, 5 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4, in a Potter-Elvehjem tissue homogenizer. Homogenized tissues were centrifuged at 15,000g for 15 min at 4°C. The supernatants were further spun at 100,000g for 1 h at 4°C and the pellets containing the membrane fraction were sonicated and resuspended in ice-cold phosphate-buffered saline, pH 7.4.
Isolation of Apical Membrane Fraction of the Rat
Syncytiotrophoblast. Apical membrane fraction of the rat placentas was
isolated using the method described by Malandro et al.
(1996) with slight
modifications. Briefly, the total membrane fraction was resuspended in
Tris-mannitol buffer (300 mM mannitol, 2 mM Tris base, pH 7.0) and homogenized
in a glass homogenizer with Teflon pestle. MgCl2 was added to a
final concentration of 10 mM. Then membranes were again homogenized, incubated
for 10 min at room temperature, and centrifuged at 2200g for 12 min.
The pellet was discarded and supernatant was centrifuged at 100,000g
for 60 min to pellet the apical-enriched membrane vesicles. The membrane
fractions were resuspended in HEPES-sucrose buffer (300 mM sucrose, 10 mM
HEPES-Tris base, pH 7.4) and frozen at -74°C.
Isolation of Basal (basolateral) Membrane Fraction of the Rat
Syncytiotrophoblast. The basal membrane fraction of the rat
syncytiotrophoblast was isolated according to the method of Malandro et al.
(1996). Bicinchoninic acid
protein assay reagent kit (Pierce Chemical, Rockford, IL) was used to
determine the protein content in the samples. The purity of membrane fractions
was analyzed by determination of alkaline phosphatase activity (the marker
enzyme of the apical membrane) and Ca2+-ATPase (the
marker enzymes of the basolateral membrane, according to the method of
Malandro et al., 1996).
Alkaline Phosphate Activity Assay. Apical and total membrane fractions were dissolved in glycine buffer (0.1 M glycine, 1 mM MgCl2, 1 mM ZnCl2, pH 10) to final protein concentration of 500 µg/ml. 4-Nitrophenyl phosphate disodium salt was added to a final concentration of 0.75 mg/ml. The mixture was incubated for 20 min at 37°C and then absorbance was measured at 410 nm (Helios gamma spectrophotometer; Thermo Spectronic, Rochester, NY).
Western Blotting of P-glycoprotein in the Rat Placenta. The Western blotting analysis was performed to detect P-glycoprotein in the rat placenta membrane fractions. Membrane fractions were suspended in equal volumes of Laemmli sample buffer, and 20 µg of protein per lane was resolved by 7% polyacrylamide SDS-PAGE gel (50 mA) and eletrotransferred (25 V, 300 mA) to nitrocellulose Hybond membranes (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). The nitrocellulose membranes were blocked in 5% blocking solution (Amersham Biosciences UK, Ltd.) and incubated in Tris-buffered saline-Tween 0.01% buffer with murine monoclonal antibody (mAb) C219 (Signet Laboratories, Dedham, MA) diluted 1:500, or with hamster mAb Ab-2/F4 (p170/P-glycoprotein/MDR Ab-2; LabVision Neomarkers, Fremont, CA). The anti-mouse secondary horseradish peroxidase-conjugated antibody (1:1000 dilution) and ECL Western blotting detection kit (both Amersham Biosciences UK, Ltd.) were used for autoradiographic detection of P-gp on FOMA blue medical X-ray films (Foma Bohemia A.S, Hradec Králové, Czech Republic). Densitometric analysis was performed using a high-resolution scanner HP 5400c (Hewlett Packard, Palo Alto, CA) and LabImage gel densitometric software version. 2.62 (Kaplan GmbH, Halle, Germany).
Immunohistochemical Localization of P-gp in the Rat Placenta. Specimens of the placentas were fixed in 4% paraformaldehyde (pH 7.35) or in Bouin's fixative fluid and then were paraffinembedded. Sections of placentas (thickness, 5 µm) were mounted on an object slide, dewaxed, and rehydrated through a series of ethanol solutions. Endogenous peroxidase activity was blocked with 3% H2O2 in 50% methanol solution for 20 min. For heat-induced antigen retrieval, the slides were boiled in 0.1 M Tris-HCl buffer, pH 1.5, for 15 min in a microwave oven at 750 W. Blocking of nonspecific background staining was performed with 10% normal goat serum (Sigma Chemie, Steinheim, Germany) in phosphate-buffered saline (PBS) solution (pH 7.4) for 30 min. Slides were incubated with primary antibody for P-glycoprotein (C219; Signet Laboratories) diluted 1:50 in PBS solution for 15 to 18 h at 4°C. After a PBS rinse, the slides were developed with the secondary antibody goat anti-mouse Ig conjugated to peroxidase-labeled polymer (DAKO EnVision+ ready-to-use; DAKO, Carpinteria, CA). Secondary antibodies were visualized with diaminobenzidine (DAKO DAB substrate-chromogen solution; DAKO) and hematoxylin counterstained. As a control for background staining, control slides were treated in the same manner, except PBS solution was substituted for the primary antibody to P-glycoprotein. Slides were examined using computer image analysis (Hund h500 light microscope, Helmut Hund, Wetzlar, Germany; JVC TK C1380E color video camera, JVC, Tokyo, Japan; LUCIA, version 4.61software, Laboratory Imaging Prague, spol. s r. o., Prague, Czech Republic).
Examination of P-gp Genes Expression by RT-PCR Method. Rat placentas
were collected into liquid nitrogen and homogenized in TRIzol reagent
(Invitrogen, Carlsbad, CA) using 1 ml of TRIzol/50 mg of tissue. Total RNA was
extracted from the tissue homogenate according to the manufacturer's
instructions. RNA integrity was assessed by electrophoresis on a 1% agarose
gel. First-strand cDNA was prepared from 1 µg of total RNA with AMV
transcriptase (Finnzymes Oy, Espoo, Finland) using oligo(dT) primer under
conditions recommended by the manufacturer. cDNA prepared from 3 ng of total
RNA was amplified by PCR with HotStar Taq polymerase (QIAGEN,
Valencia, CA); 3 mM MgCl2, 0.2 mM dNTP, 0.3 µM each primer, 0.03
U/µl polymerase; 95°C for 15 min followed by 35 cycles of 95°C for
30 s, 54°C for 1 min, 72°C for 1 min. The sequences of antisense
primers were identical for both mdr1a and mdr1b isoforms:
5'-AGCATTTCTGTATGGTATCTGCAAGC-3'. Sequence of mdr1a sense
primer was 5'-CTGCTCAAGTGAAAGGGGCTACA-3' (product length 329 bp)
and mdr1b sense primer 5'-CGCTTCTAATGTTAAAGGGGCTATG-3'
(product length 331 bp). 2-Microglobulin was used as a housekeeping gene
(B2M antisense: 5'-TACATGTCTCGGTCCCAGGTGA-3'; B2M sense:
5'-TGCCATTCAGAAAACTCCCCA-3', product length 303 bp). Amplified
segments were analyzed by electrophoresis on 1.5% agarose gel and visualized
using ethidium bromide.
Statistical Analysis. Differences between group mean values were assessed by unpaired Student's t test using STATISTICA software version. 6 (StatSoft, Tulsa, OK). Differences of p < 0.05 were considered statistically significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Effect of Maternal Inflow Concentration on the Transplacental Clearance of Rho123. The materno-fetal transplacental passage of Rho123 was found to be dependent on the maternal inflow concentration in a range of 0.42 to 4.0 µM (Fig. 1). This saturable kinetics of Rho123 indicates that transplacental passage of Rho123 is a transporter-mediated process. After fitting the data to eq. 4, Clmax describing the efficiency of P-gp to pump Rho123 back into the maternal compartment was calculated to be 0.07 ± 0.01 ml · min-1 · g-1. Clpassive diffusion was calculated to be 0.11 ± 0.01 ml · min-1 · g-1. Therefore, our data indicate that in low concentrations of Rho123 (threshold concentration <0.10 µM), P-gp could completely reverse passive diffusion of Rho123 across the barrier (Fig. 1). In higher concentrations of Rho123 (>4.0 µM), however, P-gp-mediated transport of Rho123 become saturated and is not able to return all passively diffused Rho123 back to the maternal circulation. Unfortunately, fluorometric analysis of Rho123 used in this study did not allow examining lower concentrations of Rho123 than 0.42 µM in the maternal perfusion liquid.
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Effect of CsA on Transplacental Passage of Rho123 under Nonsteady-State Conditions. Addition of 30x molar excess CsA to Rho123 solution in the maternal reservoir resulted in an increase of the materno-fetal transplacental clearance (Fig. 2A). Correspondingly, the amount of Rho123 that entered the fetal compartment rose significantly (p < 0.05) compared with controls (1.28 ± 0.43 nmol · g-1 and 2.17 ± 0.42 nmol · g-1, respectively). Doubling the amount of CsA in the maternal solution resulted in a comparable increase in the materno-fetal passage of Rho123 (data not shown). On the other hand, CsA decreased the feto-maternal clearance of Rho123 (Fig. 2B) and significantly lowered the amount of Rho123 passing from the fetal compartment into the maternal one (22.60 ± 2.18 and 19.47 ± 2.54 nmol · g-1, respectively).
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In control experiments with Rho123 (0.65 µM), its materno-fetal clearances were rising to reach a plateau (steady state) from minute 15 of the experiment (data not shown). In the case of feto-maternal passage of Rho123, steady state was reached within 10 min of the experiments (Fig. 2B). The plateau phase of the transplacental passage of Rho123 enabled us to perform experiments under steady-state conditions (see the following paragraph).
Effect of CsA, PSC833, QND, CPZ, and Sodium Azide on Transplacental
Passage of Rho123 in the Steady-State Experiments. Materno-fetal
transplacental clearances of Rho123 were nearly constant during the control
experiments (Fig. 3). An
addition of PSC833, CsA, QND, or CPZ into the maternal reservoir in the 12th
min of the experiment to reach a concentration of 10 µM (PSC833 and CsA) or
40 µM (QND and CPZ), resulted in an increase of materno-fetal
transplacental passage of Rho123 (Table
1 and Fig. 3; data
for CPZ are not shown). The most significant effect on the materno-fetal
transplacental passage of Rho123 was observed in the case of PSC833 followed
by QND and CsA (Table 1). On
the contrary, in experiments where fetomaternal transplacental passage of
Rho123 was examined, PSC833, CsA, and QND decreased feto-maternal passage of
Rho123 (Table 1 and
Fig. 3). QND had the most
potent effect on the Rho123 feto-maternal transplacental passage followed by
CsA and PSC833 (Table 1). These
data indicate that inhibitors of P-gp accelerate materno-fetal passage of
Rho123; on the other hand, inhibition of P-gp decreases the feto-maternal
passage of Rho123. This corresponds well with the same phenomenon observed in
epithelial cell lines cultured in monolayers (placental BeWo, Caco-2, etc.),
where the inhibition of a one-way activity of P-gp increases the
apical-to-basal and decreases the basal-to-apical transport of P-gp substrates
(Yumoto et al., 1999;
van der Sandt et al.,
2000).
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Apart from P-gp specific inhibitors, an ATP-synthesis inhibitor sodium azide was used to study the influence of ATP depletion on the transplacental passage of Rho123. Sodium azide affected the passage of Rho123 across the rat placenta supporting the hypothesis that P-glycoprotein, an ATP-dependent transporter, is involved in the regulation of the passage of Rho123 across the rat placenta (Fig. 4). The impact of sodium azide was comparable with that of PSC833, which is supposed to be one of the most potent specific inhibitors of P-gp (Table 1). On the other hand, the effect of sodium azide on the feto-maternal transplacental passage of Rho123 was not significant (Table 1; Fig. 4).
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Comparison of Materno-Fetal and Feto-Maternal Passages of Rho123.
Feto-maternal clearances of Rho123 were found to be significantly higher
(p < 0.01) than clearances in the opposite direction both in
steady-state and nonsteady-state experiments
(Fig. 5). PSC833 and CsA,
however, were able to partly annul this asymmetry between the materno-fetal
and feto-maternal passages of Rho123 (Table
2 and Fig. 3).
Thus, our results show that the passage of Rho123 across the intact rat
placental barrier is asymmetric due to the activity of P-gp similarly as in
the case of in vitro cultures of epithelial cell lines (intestinal Caco-2,
Yumoto et al., 1999; kidney
LLC-PK1:MDR1, van der Sandt et al.,
2000). Unlike the placental BeWo epithelial cell line, where
inhibition of P-gp lead to the same materno-fetal and fetomaternal passages of
P-gp substrates (Ushigome et al.,
2000), we did not observe a complete annulment of P-gp function by
inhibitors in the intact rat placenta. This discrepancy suggests that
transplacental passage across the intact placental barrier is a more complex
process in comparison with cellular epithelial models.
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Western Blotting of P-glycoprotein in Placental Membrane Fractions.
P-glycoprotein (molecular mass 150 kDa) was detected in the placental membrane
fractions using C219 and Ab-2/F4 monoclonal antibodies
(Fig. 6). Monoclonal antibody
Ab-2/F4 immunoreacts with P-glycoproteins encoded by human (MDR1) and
rat (mdr1a, mdr1b) genes (Huang et
al., 2001); mAb C219 shows additional immunoreactivity with
mdr2 and sister of P-glycoprotein gene products. The highest
levels of P-glycoprotein were found in the apical membrane fraction of the rat
syncytiotrophoblast (enrichment for alkaline phosphatase activity, 10.40
± 0.49). On the other hand, the basal membrane fractions showed weak
signal for P-glycoprotein.
|
Apical and basal membrane isolation methods used in this article (see
Materials and Methods) have been found to yield the apical membrane
fraction of the rat syncytiotrophoblast layer II and the basal membrane
fraction of the rat syncytiotrophoblast layer III
(Novak et al., 1997). Thus, we
suggest that P-glycoprotein is localized predominantly in the apical membrane
of the layer II, which forms crucial part of the rat materno-fetal placental
barrier.
Immunohistochemical Localization of P-gp in the Rat Placenta.
Antigen retrieval immunohistochemistry was performed for localization of P-gp
in the rat placenta using mAb C219. The rat chorioallantoic placenta is
composed of two distinct zones, junctional and labyrinthine. Strong
immunoreactivity of P-glycoprotein was observed only in the inner layers
(second or third layer) of the syncytiotrophoblast of the labyrinth zone
(Fig. 7). This well corresponds
with the fact that the labyrinth zone is thought to be the exchange area of
nutriments and drugs between mother and fetuses (the placental
"barrier"). Surprisingly, the brush-border membrane of the first
syncytiotrophoblast layer, which directly faces maternal blood, was not
immunostained as observed in the human placenta
(Nakamura et al., 1997;
Lankas et al., 1998). Fetal
capillaries of the labyrinth zone were without any immunoreactivity.
Spongiotrophoblast cells of the rat placental junctional zone, which are
important in placental hormone production, were found negative (data not
shown). These data further support the hypothesis that Pglycoprotein is
present especially in the apical membrane of the second syncytiotrophoblast
layer of the rat term placenta.
|
Expression of Genes Encoding P-gp in the Rat Placenta. Both mdr1a and mdr1b gene expressions were determined in the rat term placenta by RT-PCR analysis (Fig. 8). We detect mRNAs of both isoforms of mdr1 genes encoding rat P-glycoprotein in the rat placentas on the 22nd day of gestation. Our preliminary results show that expression of mbr1b gene could be dominant in the rat term placenta. Nevertheless, confirmation of these data using real-time RT-PCR method is in progress.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The present study examines the functional activity of P-gp in the intact
chorioallantoic rat placenta. Both the rat and human placentas are of the
hemochorial type, thus the placental barriers of both species are very similar
from the morphological and histological points of view. A recent study
employing the human trophoblast BeWo cell line brought a new view on the
function of P-gp in the placental trophoblast, suggesting that 1) the passage
of P-gp substrates is different in the apical-to-basolateral and the
basolateral-to-apical directions and that 2) P-gp accelerates the passage of
some substrates in the basolateral-to-apical direction
(Ushigome et al., 2000). On
the basis of the in vitro data achieved using BeWo cells, we speculated that,
in addition to regulating the materno-fetal passage, P-gp could also stimulate
the elimination of its substrates from the fetal circulation in the
feto-maternal direction. To confirm this speculation, the pharmacokinetics of
Rho123 across the dually perfused rat placenta in situ was investigated.
Rhodamine 123, a fluorescent dye, is a well established model compound for the
evaluation of the transport activity of P-gp in different sites of the body
and for testing of tumor cells for MDR mediated by P-gp
(Ludescher et al., 1992;
Masereeuw et al., 1997;
de Lange et al., 1998;
Yumoto et al., 1999;
van der Sandt et al., 2000).
Nevertheless, some authors suggest that a rOCT could also participate in
Rho123 transport (Masereeuw et al.,
1997; van der Sandt et al.,
2000). To assess the permeability of the intact placental barrier
for Rho123, transplacental passage of Rho123 across the placenta was expressed
as the transplacental clearance per the wet weight of the placenta
(milliliters per minute per gram). Similarly to the permeability coefficient
calculated in in vitro epithelial studies, clearance is a constant that
characterizes the ability of a drug to pass through the barrier in the in vivo
experiments.
The materno-fetal transplacental passage of Rho123 did not show the
characteristics of linear pharmacokinetics, which suggests involvement of a
transport process different from passive transport mechanisms
(Fig. 1). The same saturation
process was found in the case of CsA in the perfused rat placenta, but not in
the case of L-[3H]glucose, a marker of passive transport
(Pavek et al., 2001). One can
speculate that in very low concentrations, P-gp completely returns its
substrates back into the maternal compartment. In higher concentrations,
however, P-gp becomes saturated and drugs may pass the barrier by passive
transport. Under the steady-state conditions, the transplacental clearance of
Rho123 was found to be 8.48 times higher in the feto-maternal than in the
materno-fetal direction (Table
2; Fig. 3). In BeWo
cell monolayers, the transepithelial passages of other substrates of P-gp,
such as vinblastine, vincristine, and digoxin, were found to be 6.2-, 3.7-,
and 5.0-fold higher, respectively, in the basolateral-to-apical direction than
those in the opposite direction (Ushigome
et al., 2000). Thus, the asymmetry is higher in the rat placenta
compared with the BeWo model. We found that inhibitors of P-gp, such as
PSC833, CsA, and CZP, were able to increase significantly the materno-fetal
transplacental passage of Rho123 (Fig.
3 and Table 1). In
addition, PSC833 and QND significantly decreased the fetomaternal passage of
Rho123 across the placental barrier (Fig.
2 and Table 1).
Surprisingly, the inhibitors had less influence on the feto-maternal transport
than on the opposite one (Fig.
3 and Table 1). In
BeWo cultures, CsA was able to completely cancel the asymmetry of passage,
resulting in the same apical-to-basolateral and basolateral-to-apical
transport of three examined model compounds
(Ushigome et al., 2000). In
the intact rat placental barrier, however, P-gp inhibitors did not lead to a
complete loss of P-gp function. The feto-maternal clearance of Rho123 was
found to be higher than the materno-fetal one even in the last intervals of
the experiments. The discrepancies between the data obtained from BeWo cell
line and those from the perfused rat placenta demonstrate that the
transplacental passage across the intact placental barrier is a more complex
process. Other factors, such as presence of additional transport mechanisms,
plasma and/or tissue binding, pressures and flows of the perfusion solutions,
etc., can influence the transplacental passage of Rho123 across the intact
placenta. Despite the fact that syncytiotrophoblast is thought to be the
principal barrier component, it seems plausible that others layers, such as
endothelia of fetal capillaries, cytotrophoblast cells, and basal laminas,
could partly influence transplacental passage of Rho123 in the intact
placenta. Because P-gp showed lower effect on the feto-maternal passage of
Rho123 in comparison with the materno-fetal passage of Rho123
(Table 1), passive diffusion
seems to be important transport mechanism of Rho123 across the membranes in
the feto-maternal direction as originally suggested by Stein
(1997) in MDR-resistant cell
lines. QND influenced the passage of Rho123 to the same extent as specific and
highly potent inhibitors of P-gp, such as PSC833 and CsA. Because QND also
inhibits rOCT (Pritchard and Miller,
1993), our results suggest that there could be a coinvolvement of
an rOCT transporter in the regulation of the transplacental passage of Rho123.
P-gp and rOCT1 may provide parallel transport mechanisms in the rat placenta
similarly as it was reported in the kidney
(Miller, 1995). Sodium azide
also increased the materno-fetal passage of Rho123. Thus, ATP-depletion could
result in abolition of P-gp protective function and in an increased passage of
Rho123 across the placental barrier in the materno-fetal direction. Similar
observations have been found in Caco-2 epithelial cell model
(Augustijns et al., 1993).
Based on these findings, we can speculate that placental hypoxia and placental
ATP depletion could result in a decreased function of P-gp, and consequently
in a higher exposure of human fetuses to some lipophilic xenobiotics from the
maternal circulation. This speculation further emphasizes the importance of
studying the functional activity of P-gp in the placental barrier.
To exclude possible interference of Rho123 metabolism with the examined
transplacental passage of Rho123, HPLC method was used to determine possible
biotransformation of Rho123 in the rat placental barrier. As reported by
Sweatman et al. (1990),
metabolites of Rho123, such as Rho110 (deacylated metabolite of Rho123) and/or
its glucuronide conjugate, are formed in the rat. We found that only
negligible amount of Rho123 passing the rat placenta was metabolized into
Rho110, and therefore metabolism of Rho123 did not interfere with the study of
P-gp-mediated transport processes.
To detect and localize P-gp in the rat placentas, immunochemical and
immunohistochemical methods using mAb C219 and Ab-2/F4 have been used. As
opposed to the human placental barrier that comprises one syncytiotrophoblast
layer, there are three trophoblastic layers in the rat placental labyrinth;
two of them are thought to be syncytium (layer II and III). Layer I facing
maternal blood is of cellular nature with numerous fenestrations without
diaphragmata and thus do not represent a barrier to small compounds (Metz et
al., 1976,
1978). Syncytial layers II and
III are connected each other by numerous gap junction channels composed
preferentially of connexin26 (Metz et al.,
1976; Shin et al.,
1996). The gap junctions enable exchange of small watersoluble
substances, e.g., glucose, between syncytiotrophoblast layers as suggested by
Shin et al. (1997) and as was
clearly demonstrated by Gabriel et al.
(1998) in connexin26-defective
mice. We found the highest levels of P-gp predominantly in the apical
membranes of the second syncytiotrophoblast layer, which is considered the
"barrier" layer of the rat chorioallantoic placenta to maternal
blood (Metz et al., 1978;
Enders and Blankenship, 1999).
Review on both human and rat placental histology and morphology has been
published recently by Enders and Blankenship
(1999). This localization of
P-glycoprotein corresponds well with the function of the membrane. Continuous
location of the P-gp in the apical membrane of the syncytiotrophoblast layer
II suggests that P-gp is strategically located to face the incoming
xenobiotics from the maternal blood before they enter fetal vessels and other
fetal tissues. Our results also suggest that both mdr1a and
mdr1b genes encoding rat P-glycoprotein are expressed in the rat-term
placenta (Fig. 8). Our
preliminary results show that mbr1b gene product could prevail over
mdr1a, however, we are currently using real-time-RT-PCR method to
confirm the observation.
We conclude that our results support the hypothesis that P-gp contributes to the barrier function of the placenta. In addition, P-gp was found to accelerate the feto-maternal elimination of its substrates. Both these P-gp functions contribute simultaneously to the protection of the fetus against toxic injury. Consequently, P-gp inhibitors could increase the amount of substrates entering the fetus. Together, the present study emphasizes the importance of assessing the functional activity of P-gp in the human placental barrier since this knowledge could have important toxicological and therapeutic implications in pregnancy.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: P-gp, P-glycoprotein; MDR, multidrug resistance; Rho123, rhodamine 123; rOCT, rat organic cation transporter; RT-PCR, reverse transcription-polymerase chain reaction; CsA, cyclosporine; QND, quinidine; bp, base pair(s); CPZ, chlorpromazine; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody.
Address correspondence to: Dr. Petr Pavek, Department of Pharmacology and Toxicology, Charles University in Prague, Faculty of Pharmacy, Heyrovského 1203, Hradec Králové, CZ-500 05, Czech Republic. E-mail: pavek{at}faf.cuni.cz
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,
control). Inhibitors were added into the maternal solution at the 12th min of
experiments to reach 10 µM concentration of CsA (A) and PSC833 (B) or 40
µM concentration of QND (C) (
). In experiments on the feto-maternal
passage, Rho123 was present in the fetal reservoir in a concentration of 0.65
µM (
, control). Inhibitors were added into the maternal solution at
the 12th min of experiments in the same concentrations as in the materno-fetal
study (
). Data are expressed as means ± S.D. of at least four
experiments.
