Examination of the Functional Activity of P-glycoprotein in the Rat Placental Barrier Using Rhodamine 123

Abstract

Rhodamine 123 (Rho123), a model substrate of P-glycoprotein (P-gp), was used to evaluate the functional activity of P-gp efflux transporter in the rat placental barrier. The dually perfused rat-term placenta method was used. In our experiments, the materno-fetal transplacental passage of Rho123 did not meet the criteria of the first-order pharmacokinetics, suggesting an involvement of transporter-mediated process. Inhibitors of P-gp, such as [3Ј-keto-Bmt 1 ]-[Val 2 ]-cyclosporine (PSC833), cyclosporine (CsA), quinidine, and chlorpromazine, increased significantly the materno-fetal transplacental passage of Rho123 in the experiments under steady-state conditions. On the other hand, PSC833, CsA, and quinidine decreased the feto-maternal passage of Rho123. Similarly, in the experiments carried out under nonsteady-state conditions, CsA accelerated the passage of Rho123 in the materno-fetal direction and decreased its passage in the opposite direction. Feto-maternal transplacental clearances of Rho123 were found to be considerably higher than those in the materno-fetal course. Potent P-gp inhibitors, such as PSC833 or CsA, partially canceled the asymmetry. Negligible metabolism of Rho123 into its major demethylated metabolite rhodamine 110 was observed in the rat placenta. Expression of P-gp genes was detected using immunohistochemical, Western blotting, and reverse transcription-polymerase chain reaction methods preferentially in the second rat syncytiotrophoblast layer. In conclusion, these data suggest that P-gp limits the entry of Rho123 into fetuses and at the same time it accelerates the feto-maternal elimination of the model compound. Therefore, it seems plausible that pharmacokinetics of xenobiotics in the rat placental barrier could be controlled by P-gp in both directions.

Multidrug resistance protein P-glycoprotein does not recognize nanoparticle C60: experiment and modeling

“…Rho123, as a widely used cationic fluorescent dye, is a well established model substrate for testing the functional transport of P-gp. [34][35][36] Diluted suspensions of the K562S and K562R cells (ca.1 Â 10 7 ) were mixed with the fluorescent solution to reach a final concentration of 0.5, 1.5, 2, 5 mg mL À1 of Rho123 37 and incubated at 37 C for 2 h, respectively. The results of the FCM analysis in Fig.…”

Expression and Transport Activity of Breast Cancer Resistance Protein (Bcrp/Abcg2) in Dually Perfused Rat Placenta and HRP-1 Cell Line

“…Preparation of rat placental tissue was performed as described previously (Pavek et al, 2003). The antigen (Bcrp) was unmasked by heating the specimens in sodium citrate buffer, pH 6.0, two times for 8 min each in a microwave oven at 750 W. Slides were incubated with a polyclonal primary antibody for BCRP (M-70; Santa Cruz Biotechnology, Inc.) diluted 1:10 in bovine serum albumin for 15 to 18 h at 4°C.…”

“…The method of dually perfused rat placenta was used as described previously (Pavek et al, 2003). In brief, one uterine horn was excised and submerged in heated Ringer's saline.…”

“…The dually perfused rat placenta, in particular, is a well established model that has been successfully used to investigate placental physiology and pharmacology (Stulc et al, 1995;Kertschanska et al, 2000). In our previous studies, we used this experimental model to evaluate functional activity of P-gp in the rat placenta (Pavek et al, 2001(Pavek et al, , 2003.…”

Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1) Is Required for Branching Morphogenesis in the Chorioallantoic Placenta

“…At high concentrations, this dye shows passive mother-to-fetus passage (31). This dye is also a model substrate for P-glycoprotein present in syncytiotrophoblasts of the labyrinth layer, reflecting feto-maternal transplacental clearances (31).…”