Efﬂux Protein Expression in Human Retinal Pigment Epithelium Cell Lines
and Arto Urtti
Received December 2, 2008; accepted April 2, 2009; published online April 21, 2009
Purpose. The objective of this study was to characterize efﬂux proteins (P-glycoprotein (P-gp), multidrug
resistance proteins (MRP1–6) and breast cancer resistance protein (BCRP)) of retinal pigment
epithelium (RPE) cell lines.
Methods. Expression of efﬂux proteins in two secondary (ARPE-19, D407) and two primary (HRPEpiC and
bovine) RPE cell lines was measured by quantitative RT-PCR and western blotting. Furthermore, activity of
MRP1 and MRP5 of ARPE-19 cell line was assessed with calcein-AM and carboxydichloroﬂuorescein
Results. Similar efﬂux protein proﬁle was shared between ARPE-19 and primary RPE cells, whereas D407
cell line was notably different. D407 cells expressed MRP2 and BCRP, which were absent in other cell lines
and furthermore higher MRP3 transcript expression was found. MRP1, MRP4 and MRP5 were identiﬁed
from all human RPE cell lines and MRP6 was not expressed in any cell lines. The pattern of efﬂux protein
expression did not change when ARPE-19 cells were differentiated on ﬁlters. The calcein-AM and CDCF
efﬂux tests provided evidence supporting MRP1 and MRP5 activity in ARPE-19 cells.
Conclusions. MRP1, MRP4 and MRP5 are the main efﬂux transporters in RPE cell lines. There are
differences in efﬂux protein expression between RPE cell lines.
KEY WORDS: blood retinal barrier; cell model; drug transport; efﬂux proteins; retinal pigment
Drug delivery to the retina and vitreous remains a
challenge in ophthalmology because the posterior part of
the eye is well protected by blood–retinal barrier (BRB).
BRB is similar to blood brain barrier (BBB) that protects the
brain from harmful and toxic substances, but also restricts
drug permeation (1). BRB consists of the endothelium of the
retinal vessels (the inner barrier) and the retinal pigment
epithelium (RPE, the outer barrier) (2). Based on current
information it is not possible to draw conclusions regarding
the relative quantitative importance of the inner and outer
parts of BRB (3).
RPE protects the neural retina from the systemically
circulating xenobiotics. Systemic drug delivery to the poste-
rior eye segment is feasible only for drugs with a wide
therapeutic index, like some antibiotics. Systemic adverse
effects prevent this approach in most cases. Therefore, retinal
diseases are often medicated by invasive intravitreal injec-
tions that result in adequate drug concentrations. The recent
introduction of antibodies and aptamer to treat the exudative
form of age-related macular degeneration has increased the
use of intravitreal injections signiﬁcantly thereby increasing
the risk of complications. Periocular and subconjunctival drug
administration is an alternative to intravitreal injections (4),
but the drug must cross the sclera and RPE to reach the
retina and vitreous. The analysis of Pitkänen et al.(5)
revealed that the RPE is equal (lipophilic small molecules)
or more important barrier (hydrophilic or large molecules)
than the sclera. Finally, RPE plays a key role in ocular
elimination of drugs after intravitreal administration (6).
Both physical (tight junctions, cell membranes) and dynamic
(e.g. transporters) factors determine the permeability of
RPE that has inﬂuence on drug delivery, efﬁcacy, and
toxicity after intravitreal, subconjunctival, periocular and
As in several other barriers (e.g. blood brain barrier,
placenta), the membrane transporters may confer barrier
selectivity to the BRB (3). Transporters show usually
polarized distribution in the cell membranes and they are
classiﬁed into inﬂux and efﬂux transporters depending on the
direction of transport. Efﬂux proteins of ATP-binding cas-
sette (ABC) family pump drugs from the cells to extracellular
space (7). P-glycoprotein (P-gp, ABCB1 encoded by MDR1
gene) which was ﬁrst identiﬁed in cancer tissue confers
resistance to various drugs including cancer therapeutics,
antibiotics, corticoids, and immunosuppressive agents (7,8).
2009 Springer Science + Business Media, LLC
Pharmaceutical Research, Vol. 26, No. 7, July 2009 (
Department of Pharmaceutics, University of Kuopio, Yliopiston-
ranta 1C, 70210, Kuopio, Finland.
Centre for Drug Research, University of Helsinki, Helsinki, Finland.
Division of Biopharmacy and Pharmacokinetics, University of
Helsinki, Helsinki, Finland.
Department of Ophthalmology, University of Kuopio, Kuopio,
To whom correspondence should be addressed. (e-mail: Eliisa.