Biochemical and Biophysical Research Communications
Effects of maturation on RNA transcription and protein expression of four MRP genes in human placenta and in BeWo cells
Section snippets
Materials and methods
Placental tissues and cell cultures. Normal human placentas were obtained at term from healthy pregnancies and in the first trimester (9–10 weeks of gestation) from therapeutic interruptions of pregnancy. These were kindly provided by Gynecology and Obstetric Department of “Ospedale Infantile Burlo Garofolo,” Trieste, Italy, with a protocol approved by the Ethical Committee of the University of Trieste.
BeWo cells were obtained from Istituto Zooprofilattico Sperimentale (Brescia, Italy) and cell
Quantitative PCR
The melting dissociation profiles (data not shown), performed on MRP1, MRP2, MRP3, and MRP5 cDNAs, allowed confirmation of the specificity of the amplifications. The melting temperatures of MRP1, MRP2, MRP3, and MRP5 amplicons were 86.9±0.2, 86.1±0.1, 83.3±0.4, and , respectively, whereas GAPDH had a melting temperature of . The specificity of the amplification was also confirmed by direct sequencing and sequence alignment with the MRP1, MRP2, MRP3, and MRP5 sequences
Discussion
During gestation, it is essential that the development of the placenta is synchronized with that of the embryo. The placental barrier, which interfaces between the fetus and the mother, must cope with the gradually increasing requirements of the fetus for supply of nutrients and removal of toxic metabolites. At the different stages of fetal development, these functions must be served by a constantly evolving differential expression of the various genes involved in the relevant transplacental
Acknowledgements
Authors are deeply grateful to Dr. J.D. Ostrow for critical reading of the manuscript. L.P. and C.F. were supported in part by career development awards from Bracco SpA, Italy; D.P. was supported by a long term fellowship from University of Trieste. This work was also supported by IRCCS Burlo Garofolo Grant RF98/67, by grants from the Italian Ministry of Education (MIUR, Rome, Italy) and Fondo Studi Fegato of Trieste, Italy (FCRT-00-02).
References (36)
- et al.
Glutamine transport in human and rat placenta
Placenta
(1997) - et al.
The multidrug resistance protein family
Biochim. Biophys. Acta
(1999) - et al.
Mechanisms for the transport of unconjugated bilirubin in human trophoblastic BeWo cells
FEBS Lett.
(2001) - et al.
Functional characterization of l-alanine transport in a placental choriocarcinoma cell line (BeWo)
Placenta
(1994) - et al.
Preparation of an antibody recognizing both human and rodent MRP1
Biochem. Biophys. Res. Commun.
(2001) - et al.
Isolation and partial characterization of the basal cell membrane of human placental trophoblast
Biochim. Biophys. Acta
(1983) - et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987) - et al.
Analysis of the intron–exon organization of the human multidrug-resistance protein gene (MRP) and alternative splicing of its mRNA
Genomics
(1997) - et al.
cDNA cloning and inducible expression of human multidrug resistance associated protein 3 (MRP3)
FEBS Lett.
(1998) - et al.
Toxicological relevance of the multidrug resistance protein 1 MRP1 (ABCC1) and related transporters
Toxicology
(2001)
Evidence for carrier-mediated transport of unconjugated bilirubin across plasma membrane vesicles from human placental trophoblast
Placenta
Tissue distribution and induction of human multidrug resistant protein 3
Lab. Invest.
The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides
J. Biol. Chem.
Fetal bilirubin metabolism and neonatal jaundice
Placental transport function
Reprod. Fertil. Dev.
Evidence for location of the CFTR in human placental apical membrane vesicles
Am. J. Physiol.
Placental amino acid transport
Am. J. Physiol.
Interaction between cholephilic anions and bile acid transport across basal membrane of human trophoblast
Am. J. Physiol.
Cited by (85)
Interplay of drug transporters P-glycoprotein (MDR1), MRP1, OATP1A2 and OATP1B3 in passage of maraviroc across human placenta
2020, Biomedicine and PharmacotherapyRole of the efflux transporters BCRP and MRP1 in human placental bio-disposition of pravastatin
2018, Biochemical PharmacologyCitation Excerpt :Thus, the results of inhibition experiments with 100 µM of indomethacin and benzbromarone (Fig. 4) showed that the uptake of [3H]-pravastatin determined in placental vesicles prepared from apical and basal membranes is mediated by multiple efflux transporters. Taking into consideration results from western blot analysis and previously reported high mRNA expression of BCRP [34,35] especially in term placentas [11] as well as relative abundance of MRP1 compared to other MRPs [34,36], the in vitro ATP-dependent uptake of [3H]-pravastatin by membrane vesicles could be primarily attributed to BCRP and MRP1. In the range of pravastatin concentrations tested, uptake of [3H]-pravastatin by BCRP or MRP1 overexpressed vesicles showed standard saturation curves with relatively high affinity to the transporters (Fig. 5B&D).
The significance of abc transporters in human placenta for the exposure of fetus to xenobiotics
2017, Reproductive and Developmental ToxicologyDon't trust an(t)ybody - Pitfalls during investigation of candidate proteins for methylmercury transport at the placental interface
2016, PlacentaCitation Excerpt :High MRP2 levels in human placentas were shown in four studies (using monoclonal antibody clone M2III-6 [20–22] and antibody clone M2 I-4 [23]). Our data, however, are in line with two other studies, where MRP2 expression could either not be detected in crude placental membrane fractions and BeWo cells (M2III-6 [24]) or only very faint bands were seen in trophoblast membrane fractions and BeWo cells (M2 I-4; [25]). None of these studies had tested antibody specificity by e.g., protein overexpression or downregulation as recently suggested [10], and in all studies only parts of immunoblots were shown.