Functional Expression Of P-glycoprotein Research Articles

Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?

Drug deliveryto the central nervous system (CNS) is greatly limited by the blood-brain barrier (BBB). Physical and biochemical properties of the BBB have rendered treatment of CNS diseases, including those with a hypoxia/reoxygenation (H/R) component, extremely difficult. Targeting endogenous BBB transporters from the ATP-binding cassette (ABC) superfamily (i.e., P-glycoprotein (P-gp)) or from the solute carrier (SLC) family (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents)) has been suggested as a strategy that can improve delivery of drugs to the brain. With respect to P-gp, direct pharmacological inhibition using small molecules or selective regulation by targeting intracellular signaling pathways has been explored. These approaches have been largely unsuccessful due to toxicity issues and unpredictable pharmacokinetics. Therefore, our laboratory has proposed that optimization of CNS drug delivery, particularly for treatment of diseases with an H/R component, can be achieved by targeting Oatp isoforms at the BBB. As the major drug transporting Oatp isoform, Oatp1a4 has demonstrated blood-to-brain transport of substrate drugs with neuroprotective properties. Furthermore, our laboratory has shown that targeting Oatp1a4 regulation (i.e., TGF-β signaling mediated via the ALK-1 and ALK-5 transmembrane receptors) represents an opportunity to control Oatp1a4 functional expression for the purpose of delivering therapeutics to the CNS. In this review, we will discuss limitations of targeting P-gp-mediated transport activity and the advantages of targeting Oatp-mediated transport. Through this discussion, we will also provide critical information on novel approaches to improve CNS drug delivery by targeting endogenous uptake transporters expressed at the BBB.

Functional expression of P-glycoprotein in the hepatic canalicular membrane of developing rats

□ P-glycoprotein (P-gp), the multidrug resistance gene product, is expressed in a normal liver exclusively on the canalicular membrane of the hepatocyte. The objective of this study was to examine the effect of age on the P-gp transport system using canalicular membrane (cLPM) vesicles isolated from the liver of developing (22 days old) and adult rats. No differences in protein yield, intravesicular volumes, and enrichments of cLPM enzymes or enzymes representing contamination of subcellular organelles were found for vesicles isolated from both groups, demonstrating the isolation of similar cLPM vesicle preparations. The transport of daunomycin (DNM), a P-gp substrate, was used to study age-related functional differences in P-gp. DNM uptake in the presence of ATP was greater than uptake in the absence of ATP in both young and adult cLPM vesicles, showing that P-gp is functional in both groups. In young and adult groups only ATP was a potent stimulator of transport when compared with ATP degradation products and a nonhydrolyzable ATP analogue. Although ATP-dependent uptake tended to be greater in the adult compared to the young, there was no statistically significant difference in DNM kinetics (Vmax, km, γ) between groups. Canalicular membrane from the young rats showed decreased fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene; however there was no significant difference between groups. Examination of P-gp expression using the monoclonal antibody C219 revealed similar levels of expression in the young as in the adult. Our results suggest that P-gp in the bile canaliculus of developing rats is functional with similar levels of function and expression as observed in the adult.

Expression and Activity of p-Glycoprotein Elevated by Dexamethasone in Cultured Retinal Pigment Epithelium Involve Glucocorticoid Receptor and Pregnane X Receptor

To investigate whether dexamethasone has an effect on functional expression of p-glycoprotein in cultured human RPE and, if so, whether this occurs through interaction with glucocorticoid receptor (GR) and pregnane X receptor (PXR). The human RPE D407 was treated with increasing concentrations of dexamethasone and/or RU486 for various time periods up to 24 hours. Treated cells were collected for cell viability, expressions of p-glycoprotein and PXR, and rhodamine 123 accumulation assays. GR expression plasmid and rifampicin were chosen to investigate the relationship of GR/PXR activation and p-glycoprotein expression. Significant increases in p-glycoprotein, as indicated by mRNA and protein levels, as well as by functional activity, were induced within 12 hours of dexamethasone treatment, persisted as long as 24 hours, and were dose-dependent and attenuable with coculture of RU486. In parallel, a dose-dependent upregulation of PXR was notable at both mRNA and protein levels by 24 hours of dexamethasone treatment, and was partially reversible with RU486 coculture. Additionally, transfection of GR expression plasmid increased the transitional expressions of PXR and p-glycoprotein in untreated cells, and enhanced PXR transcriptional expression in dexamethasone-treated cells. Further, PXR silencing inhibited the dexamethasone-induced p-glycoprotein alterations; however, rifampicin had no apparent effects on the dexamethasone-induced p-glycoprotein alterations. Our results suggest for the first time that expression and activation of p-glycoprotein involve GR and PXR in human RPE.

High Glucose Decreases Expression and Activity of p-glycoprotein in Cultured Human Retinal Pigment Epithelium Possibly through iNOS Induction

Inhibition of p-glycoprotein under hyperglycemic conditions has been reported in various barrier tissues including blood-brain barrier, intestine, and kidney, and has been linked to significant clinical complications. However, whether this is also true for the outer blood-retinal barrier constituted by retinal pigment epithelium, or has a role in pathogenesis of diabetic retinopathy is not yet clear. In this study, using cultured human retinal pigment epithelium cell line D407, we found that high glucose exposure induced a significant decrease in p-glycoprotein expression both at mRNA and at protein levels, accompanied by an attenuated p-glycoprotein activity determined by intracellular rhodamine 123 retention. In marked contrast, the expressions of both mRNA and protein levels of inducible nitrate oxide synthase (iNOS) increased, and were accompanied by increased extracellular nitrate/nitrite production by Griess reaction. In addition, mRNA levels of nuclear receptors revealed a decreased expression of pregnane X receptor after the exposure of high glucose. However, the subsequent alterations in production of nitrate/nitrite, functional expression of p-glycoprotein, and mRNA levels of pregnane X receptor were partially blocked when pretreated with S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea•2HBr (PBITU), a selective iNOS inhibitor. Moreover, the effects of PBITU were antagonized with the addition of L-arginine, a substrate for NO synthesis. Our in vitro results suggest for the first time that iNOS induction plays a novel role in decreased p-glycoprotein expression and transport function at the human outer blood-retinal barrier under hyperglycemic conditions and further support the concept of inhibiting iNOS pathway as a therapeutic strategy for diabetic retinopathy.

Methodology for development of a physiological model incorporating CYP3A and P-glycoprotein for the prediction of intestinal drug absorption

The small intestine poses a major barrier to the efficient absorption of orally administered therapeutics. Intestinal epithelial cells are an extremely important site for extrahepatic clearance, primarily due to prominent P-glycoprotein-mediated active efflux and the presence of cytochrome P450s. We describe a physiologically based pharmacokinetic model which incorporates geometric variations, pH alterations and descriptions of the abundance and distribution of cytochrome 3A and P-glycoprotein along the length of the small intestine. Simulations using preclinical in vitro data for model drugs were performed to establish the influence of P-glycoprotein efflux, cytochrome 3A metabolism and passive permeability on drug available for absorption within the enterocytes. The fraction of drug escaping the enterocyte (F(G)) for 10 cytochrome 3A substrates with a range of intrinsic metabolic clearances were simulated. Following incorporation of P-glycoprotein in vitro efflux ratios all predicted F(G) values were within 20% of observed in vivo F(G). The presence of P-glycoprotein increased the level of cytochrome 3A drug metabolism by up to 12-fold in the distal intestine. F(G) was highly sensitive to changes in intrinsic metabolic clearance but less sensitive to changes in intestinal drug permeability. The model will be valuable for quantifying aspects of intestinal drug absorption and distribution.

Clinicopathologic and Radiopharmacokinetic Factors Affecting Gamma Probe–Guided Parathyroidectomy

The kinetics of technetium Tc 99m sestamibi (MIBI) in primary hyperparathyroidism are variable and affected by the cellular size of the abnormal glands, the parathyroid hormone levels, and the functional expression of P-glycoprotein (Pgp). The success of gamma probe-guided parathyroidectomy is closely related to the parathyroid-to-thyroid activity ratio at the time of surgery. Preoperative determination of maximum uptake ratio may improve the surgical outcome. Thirty-one patients with primary hyperparathyroidism attributed to a solitary parathyroid adenoma (27 patients) or multiglandular hyperplasia (4 patients) underwent dynamic MIBI imaging preoperatively. Maximum MIBI activity and activity elimination half-life in the abnormal parathyroid glands and thyroid glands were measured, and the maximum uptake ratio was calculated. After a second MIBI injection on the day of surgery, all patients underwent gamma probe-guided parathyroidectomy and cervical exploration. Timing of surgery after MIBI injection was individualized according to the optimal time to surgery (time to maximum uptake ratio), which was determined by preoperative scintigraphy. During surgery, the gamma probe was used to measure ex vivo counts of excised lesions and adjacent postexcision normal tissue (background). Image characteristics, MIBI kinetics, and gamma probe findings were correlated with gland volume, oxyphil cell content, Pgp expression, and serum parathyroid hormone levels. Probe localization of abnormal glands at maximum uptake ratio was successful in all patients. The volume of the parathyroid lesion ranged from 0.03 to 9.8 mL (median, 0.7 mL). Parathyroid maximum MIBI activity correlated with the volume of the gland (r = 0.54, P = .002) and serum parathyroid hormone level (r = 0.58, P = .001). No correlation between maximum MIBI activity and oxyphil cell content or Pgp expression could be demonstrated. Elimination half-life of MIBI from parathyroid inversely correlated with Pgp (r = -0.36, P = .05). The ex vivo lesion-background count ratio positively correlated with volume of the gland (r = 0.66, P = .001) and parathyroid hormone level (r = 0.48, P = .006). Ex vivo lesion counts and Pgp expression were negatively correlated (r = -0.37, P = .04). A strong relationship between volume of the parathyroid gland, serum parathyroid hormone levels, and MIBI uptake exists in primary hyperparathyroidism. Gamma probe-guided localization of abnormal gland(s) can be more successful if surgery is undertaken at maximum uptake ratio. High Pgp expression increases MIBI parathyroid clearance rate, decreases gamma probe counts, and may significantly alter the optimal time to surgery.

The rate of gastric emptying determines the timing but not the extent of oral tacrolimus absorption: simultaneous measurement of drug exposure and gastric emptying by carbon-14-octanoic acid breath test in stable renal allograft recipients.

Tacrolimus is characterized by a highly variable oral bioavailability and narrow therapeutic window. Tacrolimus absorption from the gastrointestinal tract is to a large extent determined by the genotypic, phenotypic, and functional expression of P-glycoprotein and CYP3A in the gut wall and liver. It is disputed whether the gastric emptying rate per se is important for determining oral bioavailability of tacrolimus and whether delayed gastric emptying is clinically relevant for therapeutic drug dosing. We conducted a pharmacokinetic study in 50 renal recipients, measuring simultaneously the rate of gastric emptying using a carbon-14-octanoic acid breath test and quantifying drug exposure by area under the concentration-time curve sampling. Gastric half emptying time (t1/2) significantly correlated with time to reach maximum blood tacrolimus (tmax) concentration (r2 = 0.30; p < 0.0001), whereas the gastric emptying coefficient, reflecting the overall gastric emptying rate, showed a weak inverse correlation with tmax (r2 = 0.14; p = 0.007). The time-dependent rate of gastric emptying strongly correlated with the simultaneously measured blood tacrolimus concentration over the first 4 h after oral drug administration (r2 = 0.96; p < 0.0001). Comparison between patients with and without delayed gastric emptying confirmed that maximum blood tacrolimus concentration was reached significantly more slowly in the former group (tmax, 2 +/- 1 h versus 1.48 +/- 0.68 h; p = 0.04), whereas the extent of tacrolimus absorption was not different. Despite a strong association between gastric emptying rate and the timing of tacrolimus absorption from the gut in stable recipients, gastric emptying rate does not affect the total extent of drug absorption and is not responsible for significant alterations in drug exposure, even in situations of delayed gastric emptying.

Cellular localization and functional expression of P‐glycoprotein in rat astrocyte cultures

We investigated the cellular/subcellular localization and functional expression of P-glycoprotein, an ATP-dependent membrane-associated efflux transporter, in astrocytes, a brain parenchyma compartment that is poorly characterized for the expression of membrane drug transporters. Analyses were carried out on primary cultures of astrocytes isolated from the cerebral cortex of neonatal Wistar rats and CTX TNA2, an immortalized rat astrocyte cell line. Both cell cultures display morphological features typical of type I astrocytes. RT-PCR analysis revealed mdr1a and mdr1b mRNA in primary cultures of astrocytes and in CTX TNA2 cells. Western blot analysis using the P-glycoprotein monoclonal C219 antibody detected a single band of appropriate size in both cell systems. Immunocytochemical analysis using the monoclonal antibodies C219 and MRK16 labeled P-glycoprotein along the plasma membrane, caveolae, coated vesicles and nuclear envelope. Immunoprecipitation studies using the caveolin-1 polyclonal H-97 antibody demonstrated that P-glycoprotein is physically associated with caveolin-1 in both cell culture systems. The accumulation of [(3)H]digoxin (an established P-glycoprotein substrate) by the astrocyte cultures was significantly enhanced in the presence of standard P-glycoprotein inhibitors and an ATP depleting agent. These results demonstrate the cellular/subcellular location and functional expression of P-glycoprotein in rat astrocytes and suggest that this glial compartment may play an important role in the regulation of drug transport in the CNS.

Molecular evidence and functional expression of P-glycoprotein (MDR1) in human and rabbit cornea and corneal epithelial cell lines.

Efflux pumps such as P-glycoprotein (P-gp; MDR1) are believed to be a major barrier to drug delivery. The purpose of this work was to determine whether cornea and corneal epithelial cells expresses the functionally active P-gp efflux pump. Cultured rabbit primary corneal epithelial cells (rPCECs) and a corneal cell line (Statens Seruminstitut rabbit cornea [SIRC] cells) were selected as the model. Rhodamine-123 (Rho-123), a P-gp substrate, was used as a P-gp probe. To confirm gene expression, RT-PCR was performed with appropriate pairs of primers for rabbit and human MDR1. Subcloning, sequencing, and protein sequence determination were performed to confirm P-gp. Permeability of [(3)H] cyclosporin A (CsA) across SIRC cells was found at 1.74 x 10(-6) cm/s in the apical-to-basolateral and 5.1 x 10(-6) cm/s in the basolateral-to-apical directions. Uptake of Rho-123 across both SIRC cells and rPCECs was time and temperature dependent. Rho-123 uptake in SIRC cells was 14.4 picomoles/mg protein and in the presence of CsA (10 micro M) was 70.8 picomoles/mg protein at 3 hours. Uptake in rPCECs was the highest at 3 hours. Western blot analysis indicated a 170-kDa band confirming the presence of P-gp. Human cornea was also checked for the presence of P-gp. RT-PCR data indicated one single band, which was subcloned and sequenced to confirm the presence of P-gp. The protein sequence deduced from the fragment product indicated more than 89% homology with human MDR1. Functional and molecular characterization showed the existence of P-gp in human cornea, rabbit cornea, and a rabbit corneal cell line. This knowledge of the existence of P-gp will help in development of better ocular drug delivery strategies.

Dexrazoxane significantly impairs the induction of doxorubicin resistance in the human leukaemia line, K562.

Dexrazoxane combined with doxorubicin (+ 5-fluorouracil + cyclophosphamide – the FAC regime) leads to a significant decrease in doxorubicin cardiotoxicity and a significant increase in median survival time for patients with advanced breast cancer responsive to FAC. The reason for this increase in survival may be due to interference with the mechanism involved in the emergence of multidrug resistance (MDR). In order to test this hypothesis, we induced resistance to doxorubicin in the K562 cell line by growing cells in increasing concentrations of doxorubicin (10–30 nM) in the presence and absence of dexrazoxane (20 nM). The doxorubicin sensitivity of all resultant sublines was measured using the MTT assay. Flow cytometry was used to assess the MDR1 phenotype, measuring P-glycoprotein expression with MRK 16 antibody and drug accumulation in the presence and absence of PSC 833 for functional P-glycoprotein. Long-term growth in doxorubicin increased the cellular resistance (IC 50) of K562 cells in a concentration-dependent manner (r2 = 0.908). Doxorubicin resistance was not induced in the presence of dexrazoxane (P< 0.0001) for several months. In parallel, the expression of functional P-glycoprotein was delayed after concomitant addition of dexrazoxane to the selecting medium (P< 0.001). Dexrazoxane did not act as a conventional modulator of P-glycoprotein. These results suggest that dexrazoxane may delay the development of MDR1, thus allowing responders to the FAC regime to continue to respond. © 2001 Cancer Research Campaign http://www.bjcancer.com

Functional expression of P-glycoprotein in primary cultures of human cytotrophoblasts and BeWo cells☆

The objective of this study was to investigate the functional expression of the efflux transporter, P-glycoprotein (P-gp), in primary cultures of human cytotrophoblasts and BeWo cell monolayers. Uptake studies with primary cultures of human cytotrophoblasts or BeWo cells were conducted with calcein-AM and vinblastine (P-gp markers) or fluorescein (MRP marker) in the presence of specific P-gp or MRP inhibitors. Results showed that the accumulation of P-gp substrates calcein-AM and vinblastine by BeWo cells or primary cultures of human cytotrophoblasts was significantly enhanced in the presence of a typical P-gp inhibitor, cyclosporin-A, or other inhibitors such as quinidine, verapamil, and dipyridamole. MRP inhibitors had no effect on the accumulation of calcein or fluorescein by BeWo cells. Western blots confirmed the presence of multidrug resistant gene product 1 (MDR1) in both primary cultures of human cytotrophoblasts and BeWo cells. This study demonstrates functional P-gp in term human trophoblasts and further supports the use of primary cultures of human cytotrophoblasts and BeWo cells as in vitro models of the trophoblast to investigate mechanisms regulating drug distribution across the placenta.

Multidrug-Resistant Phenotype Influences the Differentiation of a Human Colon Carcinoma Cell Line

The human colon carcinoma cell line HT29-D4, which constitutively expresses a very low level of the MDR1 gene product, was made multidrug resistant by transfection with a human MDR1 cDNA from the pHaMDR1/A expression vector and selection by colchicine. Resistant clones were 3- to 15-fold resistant to colchicine and were cross-resistant to doxorubicin (3- to 4-fold). MDR1 gene expression was associated with the expression of functional P-glycoprotein (gp-170); the function was reversed by verapamil and cyclosporin A. HT29-D4 cells are able to differentiatein vitroby replacement of glucose by galactose in the culture medium and also to release the carcinoembryonic antigen (CEA). Under these culture conditions, MDR1 mRNA and gp-170 were always expressed and the protein remained functional. Upon galactose treatment, resistant clones were less differentiated since they showed a heterogeneous monolayer organization accompanied by heterogeneous staining of cell-surface CEA and a high decrease (60–90%) of CEA release.

Functional Expression of P-Glycoprotein in the Hepatic Canalicular Membrane of Developing Rats

□ P-glycoprotein (P-gp), the multidrug resistance gene product, is expressed in a normal liver exclusively on the canalicular membrane of the hepatocyte. The objective of this study was to examine the effect of age on the P-gp transport system using canalicular membrane (cLPM) vesicles isolated from the liver of developing (22days old) and adult rats. No differences in protein yield, intravesicular volumes, and enrichments of cLPM enzymes or enzymes representing contamination of subcellular organelles were found for vesicles isolated from both groups, demonstrating the isolation of similar cLPM vesicle preparations. The transport of daunomycin (DNM), a P-gp substrate, was used to study age-related functional differences in P-gp. DNM uptake in the presence of ATP was greater than uptake in the absence of ATP in both young and adult cLPM vesicles, showing that P-gp is functional in both groups. In young and adult groups only ATP was a potent stimulator of transport when compared with ATP degradation products and a nonhydrolyzable ATP analogue. Although ATP-dependent uptake tended to be greater in the adult compared to the young, there was no statistically significant difference in DNM kinetics (Vmax, km, γ) between groups. Canalicular membrane from the young rats showed decreased fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene; however there was no significant difference between groups. Examination of P-gp expression using the monoclonal antibody C219 revealed similar levels of expression in the young as in the adult. Our results suggest that P-gp in the bile canaliculus of developing rats is functional with similar levels of function and expression as observed in the adult.

Failure of P-glycoprotein (MDR1) expressed in Xenopus oocytes to produce swelling-activated chloride channel activity.

1. P-glycoprotein, the protein product of the multidrug resistance (MDR1) gene, has ATP-dependent transporter activity. It has been suggested that P-glycoprotein may also function as a volume-regulated chloride channel or chloride channel regulator. To assess the chloride channel function of P-glycoprotein, we examined swelling-activated chloride conductances in Xenopus oocytes injected with human MDR1 cRNA. 2. Functional expression of P-glycoprotein in Xenopus oocytes was confirmed using Western blot analysis and by assessing transport of the P-glycoprotein substrate, calcein AM. 3. Endogenous, swelling-activated chloride conductances were virtually absent by the time P-glycoprotein expression was confirmed. Thus, this expression system afforded the advantage of assessing putative MDR1-associated chloride currents in the absence of background currents. 4. The currents activated by hypotonic shock (50%) in both MDR1-injected and control (water-injected) oocytes were not significantly different. The swelling response was due in part to the activation of a potassium-selective conductance which could be inhibited by barium. No chloride-selective currents were activated by hypotonic shock in the presence or absence of barium. Therefore, we conclude that P-glycoprotein expression does not produce a swelling-activated chloride conductance in the Xenopus oocyte expression system.

Polarized transport of docetaxel and vinblastine mediated by P-glycoprotein in human intestinal epithelial cell monolayers

The expression of the multidrug transporter P-glycoprotein has been studied in two human intestinal epithelial cell lines. No functional expression of P-glycoprotein was found in the differentiated HT29-18-C 1 cell line. The expression of P-glycoprotein in the Caco-2 cell line was very high, as judged by immunoblotting and by active efflux of vinblastine. The polarized transport of vinblastine in the basolateral to apical direction was temperature and energy dependent, and was reduced by P-glycoprotein inhibitors such as verapamil, chlorpromazine and reserpine. This adds further evidence that the polarized transport of vinblastine across Caco-2 monolayers is mediated by P-glycoprotein. The anticancer drug docetaxel (Taxotere ®) was transported in a polarized manner: basolateral to apical permeability was 20-fold higher than in the reverse direction. This polarized transport was inhibited by verapamil, chlorpromazine and reserpine, thus demonstrating that docetaxel is a substrate of P-glycoprotein. The implications of these results for the pharmacokinetics and toxicity of taxoids are discussed.

Functional expression of P-glycoprotein in Saccharomyces cerevisiae confers cellular resistance to the immunosuppressive and antifungal agent FK520.

We have recently reported that expression in yeast cells of P-glycoprotein (P-gp) encoded by the mouse multidrug resistance mdr3 gene (Mdr3) can complement a null ste6 mutation (M. Raymond, P. Gros, M. Whiteway, and D. Y. Thomas, Science 256:232-234, 1992). Here we show that Mdr3 behaves as a fully functional drug transporter in this heterologous expression system. Photolabelling experiments indicate that Mdr3 synthesized in yeast cells binds the drug analog [125I]iodoaryl azidoprazosin, this binding being competed for by vinblastine and tetraphenylphosphonium bromide, two known multidrug resistance drugs. Spheroplasts expressing wild-type Mdr3 (Ser-939) exhibit an ATP-dependent and verapamil-sensitive decreased accumulation of [3H]vinblastine as compared with spheroplasts expressing a mutant form of Mdr3 with impaired transport activity (Phe-939). Expression of Mdr3 in yeast cells can confer resistance to growth inhibition by the antifungal and immunosuppressive agent FK520, suggesting that this compound is a substrate for P-gp in yeast cells. Replacement of Ser-939 in Mdr3 by a series of amino acid substitutions is shown to modulate both the level of cellular resistance to FK520 and the mating efficiency of yeast mdr3 transformants. The effects of these mutations on the function of Mdr3 in yeast cells are similar to those observed in mammalian cells with respect to drug resistance and transport, indicating that transport of a-factor and FK520 in yeast cells is mechanistically similar to drug transport in mammalian cells. The ability of P-gp to confer cellular resistance to FK520 in yeast cells establishes a dominant phenotype that can be assayed for the positive selection of intragenic revertants of P-gp inactive mutants, an important tool for the structure-function analysis of mammalian P-gp in yeast cells.

Functional expression of P-glycoprotein in Saccharomyces cerevisiae confers cellular resistance to the immunosuppressive and antifungal agent FK520.

We have recently reported that expression in yeast cells of P-glycoprotein (P-gp) encoded by the mouse multidrug resistance mdr3 gene (Mdr3) can complement a null ste6 mutation (M. Raymond, P. Gros, M. Whiteway, and D. Y. Thomas, Science 256:232-234, 1992). Here we show that Mdr3 behaves as a fully functional drug transporter in this heterologous expression system. Photolabelling experiments indicate that Mdr3 synthesized in yeast cells binds the drug analog [125I]iodoaryl azidoprazosin, this binding being competed for by vinblastine and tetraphenylphosphonium bromide, two known multidrug resistance drugs. Spheroplasts expressing wild-type Mdr3 (Ser-939) exhibit an ATP-dependent and verapamil-sensitive decreased accumulation of [3H]vinblastine as compared with spheroplasts expressing a mutant form of Mdr3 with impaired transport activity (Phe-939). Expression of Mdr3 in yeast cells can confer resistance to growth inhibition by the antifungal and immunosuppressive agent FK520, suggesting that this compound is a substrate for P-gp in yeast cells. Replacement of Ser-939 in Mdr3 by a series of amino acid substitutions is shown to modulate both the level of cellular resistance to FK520 and the mating efficiency of yeast mdr3 transformants. The effects of these mutations on the function of Mdr3 in yeast cells are similar to those observed in mammalian cells with respect to drug resistance and transport, indicating that transport of a-factor and FK520 in yeast cells is mechanistically similar to drug transport in mammalian cells. The ability of P-gp to confer cellular resistance to FK520 in yeast cells establishes a dominant phenotype that can be assayed for the positive selection of intragenic revertants of P-gp inactive mutants, an important tool for the structure-function analysis of mammalian P-gp in yeast cells.

Functional expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells. Kinetics of vinblastine secretion and interaction with modulators

The functional expression of P-glycoprotein has been studied in confluent epithelial layers of human Caco-2 cells, a polarized, highly differentiated cell line demonstrating an intestinal absorptive cell phenotype. Expression of P-glycoprotein was localized, by indirect immunofluorescence with monoclonal antibody MRK16, to the apical brush-border, approximately 20 microns above the base of the cells. Functional, high capacity expression of P-glycoprotein in Caco-2 cell layers was demonstrated by the saturable secretion of vinblastine, a typical substrate, from basolateral to apical surfaces: Km 18.99 +/- 5.55 microM, Vmax 1285.9 +/- 281.2 pmol.cm-2 h-1. The direct correlation of apical P-glycoprotein expression with vinblastine net secretory flux was demonstrated by the reduction of this flux after treatment with MRK16 antibodies. Vinblastine secretory flux was also reduced by treatment with verapamil (R- and S-isomers with equal affinity), nifedipine, taxotere, and 1,9-dideoxyforskolin. Kinetic analyses suggest that the inhibition of vinblastine secretory flux by verapamil and nifedipine was competitive, while that by dideoxyforskolin was non-competitive, in nature. The polarized expression and activity of P-glycoprotein in Caco-2 cells is direct evidence for its secretory detoxifying function in the intestine, subserving at least one role of the gastrointestinal epithelial barrier.