The membrane transport system responsible for multidrug resistance is operating in nonresistant cells

Cultured hamster fibroblasts of the DM-15 cell line stained by rhodamine 123 gradually release the dye when placed in dye-free medium. Here we demonstrate that reserpine, verapamil, and trifluoperazine are capable of blocking this release. We also show that reserpine can inhibit the efflux of another dye, phosphine 3R, from DM-15 cells and the release of rhodamine 123 from mouse embryo fibroblasts, four mouse cell lines, and MDCK cells. The three substances that block the release of the dyes are potent inhibitors of the membrane transport system implicated in the phenomenon of multidrug resistance (MDR). By using this system MDR cells can pump many structurally unrelated drugs and dyes, including rhodamine 123 and phosphine 3R, from the cytoplasm to the outer medium. It appears from our results that the membrane transport system responsible for MDR operates slowly in nonresistant cells and can play a role in normal cell physiology.     

Characterization of rhodamine-123 as a tracer dye for use in in vitro drug transport assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.     

Assessment of P-glycoprotein-dependent drug transport in isolated rat hepatocytes using rhodamine 123

To test the activity of P-170 glycoprotein in isolated hepatocytes, a method has been developed employing the fluorescent dye rhodamine 123 (R-123). The uptake of R-123 by both freshly isolated and 4-hr-plated hepatocytes depends on dye concentration, time of incubation, and cell number. The efflux of R-123 from cells is inhibited by sodium azide and by verapamil. In standard conditions the efficiency of efflux of R-123 from cells correlates with the relative amount of immunoblottable glycoprotein. The method has been applied to detection of P-170 activity in hepatocytes from animals of different ages as well as from carcinogen-treated animals. The proposed assay appears a simple and adequate tool for the functional assessment of multidrug transporter in liver.Abbreviations AAF acetaminofluorene - MDR multidrug resistance - Pgp P-glycoprotein - R-123 rhodamine 123     

Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells.

Hematopoietic stem cells show reduced staining with a mitochondrial fluorescent dye, rhodamine 123 (Rh-123), which was supposed to indicate decreased mitochondrial activity in these cells. Rh123 and several other fluorescent dyes are substrates for transport mediated by P-glycoprotein (P-gp), an efflux pump responsible for multidrug resistance in tumor cells. We have found that staining of human bone marrow cells with fluorescent dyes is potentiated by P-gp inhibitors and inversely correlated with P-gp expression. P-gp is expressed in practically all hematopoietic progenitor cells, including long-term culture-initiating cells. The highest levels of P-gp among the progenitors are associated with cells displaying characteristics of pluripotent stem cells. These results have implications for stem cell purification and bone marrow resistance to cancer chemotherapy.     

Estradiol decreases retention of rhodamine 123 fluorescence in GH4C1 pituitary tumor cells

Rhodamine 123 is a lipophilic cationic fluorescent dye that localizes in mitochondria. We found that 17 beta-estradiol changes the ability of GH4C1 cells, clonal rat pituitary tumor cells, to retain rhodamine 123. Cells incubated with 10 micrograms/ml rhodamine 123 for 30 min at 37 C took up about equal amounts of rhodamine 123, as determined by fluorescence microscopy, regardless of whether they had been treated with estradiol. After three 5-min washes at 37 C, cells treated with 1 nM estradiol for 7 days before incubation with rhodamine 123 had lost more fluorescence than untreated cells. We further characterized the effect by flow cytometry. The difference in fluorescence between control and treated cells ranged from 50- to 500-fold. The effect of estradiol was maximal at 10(-10) M and took a week to develop fully. The effect is specific for estradiol, because estradiol and diethylstilbestrol reduced retention of rhodamine 123 fluorescence at 10(-10) M, but the same concentrations of dihydrotestosterone, progesterone, dexamethasone, and cholesterol did not. To test if the effect on rhodamine 123 fluorescence was caused by activation of the multidrug resistance transport system, we examined the effect of estradiol on the retention of daunomycin, a known substrate of the transport system. Estradiol treatment caused a 3-fold decrease in daunomycin fluorescence. We isolated clones resistant to estradiol-induced loss of rhodamine 123 fluorescence by flow cytometry and found that two clones still showed an estradiol-induced decrease in daunomycin fluorescence equivalent to that of the parent line.(ABSTRACT TRUNCATED AT 250 WORDS)     

New insights into the P-glycoprotein-mediated effluxes of rhodamines.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P-glycoprotein (P-gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, ka = VM/km, which is a measure of the efficiency of the P-gp-mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P-gp-mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P-gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P-gp-mediated efflux of rhodamine analogues in intact cells.     

Tetrandrine and fangchinoline,bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells

The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy.     

Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.     

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.     

Histone deacetylase inhibitor apicidin-mediated drug resistance: involvement of P-glycoprotein

Multidrug resistance (MDR), which is a significant impediment to the success of cancer chemotherapy, is attributable to the overexpression of membrane transport proteins, such as P-glycoprotein (P-gp), resulting in an increased drug efflux. In this study, we show that the histone deacetylase (HDAC) inhibitor apicidin leads to resistance of HeLa cells to paclitaxel through the induction of P-gp expression. Furthermore, apicidin dramatically increases the release of a fluorescent P-gp substrate, rhodamine 123, from cells. In parallel, apicidin resistance to the apoptotic potential of paclitaxel is associated with induction of P-gp expression in HeLa cells, as evidenced by specific inhibition of P-gp function using either the pharmacological inhibitor verapamil or RNA silencing. We also demonstrate the contribution of apicidin-induced functional P-gp expression to drug resistance using KB cells. Failure of P-gp induction by apicidin does not reverse paclitaxel-induced cytotoxicity in the cells. Although HDAC inhibitors are widely appreciated as a new class of anti-tumor agent, our findings clearly demonstrate that apicidin treatment may lead to P-gp-mediated resistance to other anti-tumor agents, suggesting a need for careful design of clinical applications using HDAC inhibitors.     

Retention of vital dyes correlates inversely with the multidrug-resistant phenotype of adriamycin-selected murine fibrosarcoma variants

Retention of the vital dyes rhodamine 123 (R-123) and hydroethidine (HET) correlates inversely with the multidrug resistant phenotypes of the adriamycin (ADM)-selected variants of a uv-induced murine fibrosarcoma cell line (UV-2237M). The differential affinity of these dyes for specific cellular organelles makes them unique compounds for studies of cellular transport. HET enters viable cells freely, is dehydrogenated to ethidium bromide (EtBr), and is subsequently accumulated in the nucleus. Viable cells are impermeable to extracellular EtBr, facilitating kinetic analysis of the efflux of intracellular EtBr. We found that the metabolite EtBr was rapidly cleared by ADM-resistant but not by ADM-sensitive cells. R-123 has a high affinity to mitochondria. Our results show that ADM-sensitive cells retain R-123 whereas the ADM-resistant cells do not. The clearance of both R-123 and EtBr from these cells was inhibited by verapamil. Therefore, R-123 and HET may be considered MDR-associated compounds useful in studying the MDR phenotype of cancer cells. Previously we reported a direct correlation between the level of activity of the calcium- and phospholipid-dependent protein kinase (protein kinases C) and ADM resistance in UV-2237M variant lines. In this report, we demonstrate a direct correlation between cellular calcium and MDR in these cells. Although chelation of extracellular calcium by EDTA did not alter the fluorescence profile of R-123 of the various cell lines, treating the ADM-resistant variants with verapamil restored cellular calcium to the same level as that of the parental cells and, at the same time, retarded the facilitated efflux of R-123 and EtBr and partially reversed cancer cell resistance to ADM.     

Influx and efflux kinetics of cationic dye binding to respiring mitochondria.

We have investigated the kinetics of interaction of cationic fluorescent lipophiles (dyes) rhodamine 123, rhodamine 6G, tetramethyl rhodamine ethyl ester, safranine O, 1,1'-diethyloxacarbocyanine, 1,1'-diethyloxadicarbocyanine, and 1,1'-diethylthiadicarbocyanine iodide with isolated respiring rat-liver mitochondria (RLM). Dye flux across the RLM inner membrane was measured by following the kinetics of fluorescence signal change after mixing of dye and RLM. The time course of fluorescence was analysed in terms of a kinetic model of the binding and transport processes involved. The rate constants of dye influx and efflux were extracted from the observed effect on the apparent time constant of fluorescence change to equilibrium intensity upon mixing dye with increasing concentrations of RLM. From the influx rate constants obtained, the apparent permeability constants for dye influx (at zero potential) across the membrane were calculated and ranged from 3 to 140 x 10(-4) cm/s. The influx rate constant was found to be linearly related to relative dye lipophilicity, as predicted by the model. As another test of the model, from the ratio of the influx and efflux rate constants, the apparent trans-membrane potential, psi, was calculated and found generally to agree with reported values, but to depend on the lipophilicity of the dye used. Not predicted by the simple model was a dissymmtry observed in the influx and efflux time constants for fluorescence change to equilibrium intensity. Inferences are made relating to the utility of these dyes as probes of psi.     

Membrane hyperpolarisation by valinomycin and its limitations for bacterial viability assessment using rhodamine 123 and flow cytometry

Abstract The ionophore, valinomycin, was investigated as a possible means of bacterial viability assessment using the fluorescent membrane potential dye rhodamine 123. Membrane hyperpolarisation in Escherichia coli , Pseudomonas fluorescent , Enterobacter aerogenes and Arthrobacter globiformis was examined during exponential growth and during stress by brief starvation in a high sodium, low potassium buffer using flow cytometric analysis of rhodamine 123 uptake. Dye uptake was variable both between species and amongst cells from the same culture. Exponential phase cells showed no increase in dye uptake due to valinomycin treatment. Stressed P. fluorescens cells responded to valinomycin treatment by increased dye uptake, while stressed E. coli and A. globiformis cells showed no response. Approximately 50% of stressed Eb. aerogenes cells responded to valinomycin. The results demonstrate the limitations of rhodamine dye for viability analysing the viability of diverse bacterial communities and underline the degree of cell heterogeneity in batch cultures.     

Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry

A.S. KAPRELYANTS AND D.B. KELL. 1992. The fluorescent dye rhodamine 123 (Rh 123) is concentrated by microbial cells in an uncoupler-sensitive fashion. Steady-state fluorescence measurements with Micrococcus luteus indicated that provided the added dye concentration is below approximately 1 mmol/1, uptake is fully uncoupler-sensitive and is not accompanied by significant self-quenching of the fluorescence of accumulated dye molecules. 'Viable' and 'non-viable' cells are easily and quantitatively distinguished in a flow cytometer by the extent to which they accumulate the dye. The viability of a very slowly growing chemostat culture of Mic. luteus is apparently only about40–50%, as judged by plate counts, but most of the 'non-viable' cells can be resuscitated by incubation of the culture in nutrient medium before plating. The extent to which individual cells accumulate rhodamine 123 can be rapidly assessed by flow cytometry, and reflects the three distinguishable physiological states exhibited by the culture ('non-viable', 'viable' and 'non-viable-but-resuscitable'). Gram-negative bacteria do not accumulate rhodamine 123 significantly because their outer membrane is not permeable to it; a simple treatment overcomes this. Flow cytometry using rhodamine 123 should prove of general utility for the rapid assessment of microbial viability and vitality.     

Microplate screening of the differential effects of test agents on Hoechst 33342, rhodamine 123, and rhodamine 6G accumulation in breast cancer cells that overexpress P-glycoprotein

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.     

Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.     

Evaluation of the Role of P-Glycoprotein in Ivermectin Uptake by Primary Cultures of Bovine Brain Microvessel Endothelial Cells

The P-glycoprotein efflux system located on the apical membrane of brain capillary endothelial cells functions as part of the blood-brain barrier. In this study, primary cultures of bovine brain microvessel endothelial cells (BMECs) were investigated for the presence of a P-glycoprotein system and its contribution in regulating ivermectin distribution across the blood-brain barrier. Results of rhodamine 123 uptake studies with cyclosporin A and verapamil as substrates indicated that a functional efflux system was present on BMECs. Immunoblot analysis with the C219 monoclonal antibody to the product of the multidrug resistant member 1(MDR1) gene also confirmed the expression of MDR1 in the BMECs. Unbound ivermectin was shown to significantly increase the uptake of rhodamine 123 in BMECs, however, the drug only modestly enhanced the transcellular passage of rhodamine. The results of these studies affirmed that unbound ivermectin is an inhibitor of the MDR1 efflux system in BMECs.     

Expression and functional activity of P-glycoprotein in cultured hepatocytes from Oncorhynchus mykiss

P-glycoproteins encoded by multidrug resistance 1 (mdr1) genes are ATP-dependent transporters located in the plasma membrane that mediate the extrusion of hydrophobic compounds from the cell. Using cultured isolated rainbow trout hepatocytes, we characterized an mdr1-like transport mechanism of the teleost liver. Immunoblots with the monoclonal antibody C219, which recognizes a conserved epitope of P-glycoproteins, revealed the presence of immunoreactive protein(s) of 165 kDa in trout liver and cultured hepatocytes. In trout liver sections, the immunohistochemistry with C219 stained bile canalicular structures. Compounds known to interfere with mdr1-dependent transport (verapamil, vinblastine, doxorubicin, cyclosporin A, and vanadate) all increased the accumulation of rhodamine 123 by hepatocytes. Verapamil, vinblastine, and cyclosporin A decreased the efflux of rhodamine 123 from hepatocytes preloaded with rhodamine 123. By contrast, the substrate of the canalicular cation transporter tetraethylammonium and the inhibitor of the multidrug resistance-associated protein MK571 had no effect on rhodamine 123 transport. The results demonstrate the presence of an mdr1-like transport system in the teleost liver and suggest its function in biliary excretion.     

A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell sorting

To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K₂, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl₂ before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains.     

Cmdr1, a chicken P-glycoprotein, confers multidrug resistance and interacts with estradiol

We have cloned from a chicken intestinal cDNA library Cmdr1, the first avian P-glycoprotein. Cmdr1 is 67% and 69% identical to proteins encoded by the human MDR1 and MDR2 genes, respectively. Functional expression of Cmdr1 in both mouse NIH 3T3 and yeast cells demonstrated that Cmdr1 represents the avian ortholog of human Mdr1, since it confers resistance to several anticancer drugs and the fluorescent dye rhodamine 6G. Northern and immunoblot analysis showed that CMDR1 is highly expressed throughout the intestine and in the liver, and to a considerable extent in kidney, brain, lung, heart, eye and follicles. In situ hybridization revealed a cell type-specific expression of CMDR1 in the intestinal epithelium, with high levels in the villi of the small and large intestine as well as crypt cells. These data suggest that Cmdr1 could play a role in intestinal detoxification. Most interestingly, immunoblotting showed that Cmdr1 is also expressed in ovarian tissues, particularly in theca cells, the major site for ovarian estrogen production in birds. Indeed, competition experiments indicated that Cmdr1 interacts with estradiol, since rhodamine 6G efflux was efficiently blocked by estradiol in NIH 3T3 cells expressing Cmdr1. Rhodamine efflux was also blocked by PSC-833, a specific inhibitor of steroid-transporting P-glycoproteins from mammalian cells. We propose that Cmdr1 in ovarian cells could be involved in the cell type-specific transport or release of estrogen that is essential for avian follicular development.