Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The blood-brain barrier (BBB) is a unique structure that serves to protect the brain from toxic solutes present in the systemic blood circulation as well as to allow selective access of necessary nutrients and chemical signaling molecules to the central nervous system (Tsuji and Tamai, 1998). The barrier function of BBB is due to the tight junctions formed between the brain microvessel endothelial cells, which impede paracellular diffusion of the solutes. Furthermore, the transcellular transport of many solutes is severely decreased by the drug efflux proteins, such as P-glycoprotein (Pgp), located at the luminal side of the brain microvessel endothelial cells (Seetharaman et al., 1998). As a result, the BBB presents a considerable challenge for the delivery of many pharmaceutical drugs from blood to the brain.

One emerging strategy to enhance drug delivery to the central nervous system is the coadministration of a pharmacological modulator or a formulation component that inhibits Pgp-mediated efflux of a desired therapeutic agent out of the brain (Miller and Kabanov, 1999). Both in vitro (Batrakova et al., 1998b, 1999b) and in vivo (Kabanov et al., 1989; Batrakova et al., 2001) studies demonstrated that Pluronic block copolymers can enhance the transport of solutes across the BBB. Using monolayers of polarized bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB studies demonstrated that coadministration of Pluronic P85 (P85) significantly increased transport of various Pgp substrates through inhibiting the Pgp efflux transport system (Batrakova et al., 1998b, 1999b). Furthermore, in vivo studies suggested that P85 significantly enhanced brain penetration of a Pgp substrate, digoxin, in wild-type mice expressing functional Pgp, resulting in brain/plasma levels of digoxin, similar to those observed in Pgp-deficient knock-out mice (Batrakova et al., 2001).

The practical significance of Pluronic block copolymer formulations is reinforced by the fact that these formulations have been shown to be very effective in the treatment of multiple drug-resistant tumors and are currently undergoing Phase I/IIa clinical trials for this application (Alakhov et al., 1996, 1999; Venne et al., 1996). There is overwhelming evidence that Pluronic block copolymers can inhibit the Pgp efflux system in various cells, including brain microvessel endothelial cells (Venne et al., 1996; Miller et al., 1997; Batrakova et al., 1998a,b, 1999a, 2001; Evers et al., 2000). However, the mechanism of the effect of these polymers on Pgp drug efflux system in the BBB remains unclear. Recently it was found that Pluronic block copolymer caused significant depletion in ATP levels in multidrug-resistant cells (Batrakova et al., 2000). Since Pgp-mediated efflux requires energy consumption (Hrycyna et al., 1998), this could be one reason for the inhibition of Pgp function observed with P85. On the other hand, the literature suggests that nonionic surfactants may inhibit drug efflux transport through increased membrane fluidization that induces changes in the conformation of Pgp and ATPase activity (Regev et al., 1999). The purpose of this paper is to elucidate the mechanisms of the effect of Pluronic block copolymers on the Pgp efflux system in BBB and to examine whether ATP depletion and membrane fluidization contribute to the effects of the block copolymer. To address this aim, the present work uses BBMEC monolayers as an in vitro model of BBB and human Pgp-expressing membranes for evaluation of the Pgp ATPase activity. A combination of experiments examining the kinetics, concentration dependence, and directionality of P85 effects on Pgp efflux system in BBMEC monolayers is used to uncover the mechanism of the block copolymer action.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Preparation of Pluronic Block Copolymer Solutions. The Pluronic block copolymer P85 used in this work was kindly provided by BASF Corp. (Parsippany, NJ). The solutions of P85 were prepared in assay buffer containing 122 mM sodium chloride, 25 mM sodium bicarbonate, 10 mM glucose, 10 mM HEPES, 3 mM potassium chloride, 1.2 mM magnesium sulfate, 1.4 mM calcium chloride, and 0.4 mM potassium phosphate dibasic, pH 7.4. P85 solutions were equilibrated a minimum of 1 h at 37°C before using. For microscopy studies, P85 was labeled by FITC attached to one of the block copolymer free ends as described earlier (Kabanov et al., 1992).

Cell Isolation and Culture. BBMEC were isolated from fresh cow brains using a combination of enzymatic digestion and density centrifugation as described previously (Miller et al., 1992). The cells were maintained in modified Eagle's medium [F12 culture medium supplemented with 10% horse serum, heparin sulfate (100 µg/ml), amphotericin B (2.5 µg/ml), and gentamicin (50 µg/ml)]. All tissue culture media were obtained from Invitrogen (Grand Island, NY). Isolated BBMEC were seeded at a density of 50,000 cells/cm² in 24-well plates and were used for ATP assay and (rhodamine 123) R123 accumulation studies after reaching confluence (typically within 14 days).

[³H]P85 Accumulation Studies. A tritium label was incorporated into P85 by treatment of the copolymer film with atomic tritium as previously described (Melik-Nubarov et al., 1999). The sample of [³H]P85 with specific activity of 0.3 Ci/mmol was obtained. This sample was further diluted in the solution of unlabeled P85 to obtain the desired label concentration. The accumulation of [³H]P85 was examined in confluent BBMEC monolayers at 37°C up to 90-min time intervals. For these studies, the culture medium was removed from the BBMEC monolayers and replaced with assay buffer. After 30 min of preincubation at 37°C, the assay buffer was removed and 0.5 ml of 0.5 µCi/ml (1.6 nM) [³H]P85 solution was added to the monolayers. When concentration dependence of P85 accumulation was examined, the cell monolayers were exposed for 1 h to various concentrations of [³H]P85 or 1.6 nM tritium-labeled P85 with various concentrations of unlabeled block copolymer. Then, block copolymer solutions were removed, and cells were washed three times with ice-cold PBS. BBMEC monolayers were solubilized in 1% Triton X-100 (0.5 ml), and aliquots were taken for subsequent determination of radioactivity (Tricarb 4000; Packard Instrument Co., Meriden, CT). All experiments were conducted in quadruplicate. Values for cellular accumulation of [³H]P85 were normalized for cellular protein content. Protein concentrations were determined using the Pierce (Rockford, IL) bicinchoninic acid method.

R123 Accumulation Studies. R123 accumulation in BBMEC was studied as previously described (Miller et al., 1997). Briefly, confluent cell monolayers were preincubated with the assay buffer for 30 min at 37°C, then the assay buffer was removed, and cell monolayers were exposed to 3.2 µM R123 in either assay buffer or P85 solutions for various time intervals up to 90 min. After incubation, the dye solutions were removed, and the cell monolayers were washed three times with ice-cold PBS and then solubilized in Triton X-100 (1.0%). Aliquots were removed for determination of the cellular dye using a Shimadzu RF5000 fluorescent spectrophotometer (Shimadzu, Kyoto, Japan) (_ex = 505 nm, _em = 540 nm) and cellular protein using the Pierce BCA assay. All experiments were carried out in quadruplicate.

ATP Assay. To examine the effects of the block copolymer P85 on intracellular ATP levels, the confluent BBMEC monolayers were pretreated with assay buffer for 30 min and then treated with various concentrations of P85 solutions in assay buffer for various time intervals up to 2 h. Following treatment, the cells were washed two times with ice-cold PBS, solubilized in Triton X-100 (1.0%), and frozen immediately for subsequent ATP quantification (conducted within 24 h following the sample collection). ATP was determined using a luciferin/luciferase assay (Garewal et al., 1986). For this purpose, 100-µl aliquots of cell lysate were mixed with 100 µl of ATP assay mix (FL-AAM; Sigma, St. Louis, MO). Light emission was measured with a Turner Designs luminometer (model 20/20; Sunnyvale, CA). Raw data were collected as relative light units integrated over 20 s for samples and converted to ATP concentrations with the aid of a standard calibration curve obtained using ATP standards (FL-AAS; Sigma). ATP levels were normalized for protein content, and each data point represented the mean ± S.E.M. of a minimum of four replicates.

Pgp ATPase Assay. Effects of P85 on Pgp ATPase activity were determined using suspension of membranes, expressing human Pgp (Gentest, Woburn, MA). An 0.06-ml reaction mixture containing 40 µg of membranes, 20 µl of assay buffer, with or without P85, was added to a buffer solution containing 3 to 5 mM MgATP, 50 mM Tris-MES, 2 mM EGTA, 50 mM KCl, 2 mM dithiothreitol, and 5 mM sodium azide, and incubated at 37°C for 20 min. An identical reaction mixture containing 100 µM sodium orthovanadate was assayed in parallel. Orthovanadate inhibits Pgp by trapping MgADP in the nucleotide-binding site. Thus, ATPase activity measured in the presence of orthovanadate represents non-Pgp ATPase activity and can be subtracted from the activity measured in the various samples to yield Pgp ATPase activity. The reaction was stopped by the addition of 30 µl of 10% SDS with Antifoam A (Sigma, St. Louis, MO). Aliquots (200 µl) of ammonium molybdate in 15 mM zinc acetate/10% ascorbic acid (1:4) were added to each sample and incubated for an additional 20 min at 37°C. The liberation of inorganic phosphate was detected by its absorbance at 630 nm and quantitated by comparing the absorbance to a phosphate standard curve (Druekes et al., 1995; Shepard et al., 1998).

DPH Labeling of BBMEC. DPH was used as a probe to examine the fluidity properties of the hydrocarbon region of the cell membranes (Laat et al., 1977). A suspension of BBMEC was washed twice with PBS and incubated with the 2 µM DPH labeling solution for 1 h at 37°C. Following the initial labeling with DPH, cells were washed twice with PBS to remove extracellular DPH and resuspended in an appropriate volume of PBS. To evaluate the kinetic effects of P85 in BBMEC, 30 µl of 10% P85 stock solution was added to 3 ml of cell suspension in PBS to obtain 0.1% P85 solution. Changes in membrane microviscosity were recorded immediately after addition of P85 and up to 90 min following the addition of copolymer. To examine the time frame for reversal of P85 effects on membrane fluidity, BBMEC suspension was incubated with DPH and then with P85 for 1 h at 37°C. After that, cells were washed two times with PBS to remove extracellular DPH and P85, and restitution of initial microviscosity was measured over a 2-h period.

Fluorescence Polarization Measurements. Fluorescence intensities (parallel, I, or perpendicular, I) were measured with a Hitachi F5000 spectrophotometer (Hitachi, Yokohama, Japan) equipped with a polarizer set. The fluorescence anisotropy r was calculated according to eq.1 :

(1)

Excitation wavelength was 365 nm, and emission wavelength was 425 nm. Cell suspensions were gently mixed before each reading, and readings were taken at 37°C. In all cases, corrections for stray light and intrinsic fluorescence were made by subtracting the values for I and I of unlabeled samples from those of identical but labeled samples.

(2)

1

1

Directionality of R123 Transport. Polycarbonate membrane inserts with confluent BBMEC monolayers were placed in side-by-side diffusion cells from Crown Bio Scientific, Inc. (Somerville, NJ) maintained at 37°C. Cell monolayers were preincubated for 30 min at 37°C with the assay buffer added to both donor and receiver chambers. Trans-epithelial electrical resistance values were recorded as indexes of cell viability and monolayer integrity. Under basal conditions, mean resistance was 135.0 ± 13.2  · cm². Transport of Pgp substrate R123 from apical (AP) to basolateral (BL) direction was studied. In experiments evaluating the effect of P85 applied to the apical side of BBMEC monolayers, the assay buffer in the donor chamber was replaced with R123 in either assay buffer alone or 0.1% P85 containing solution, and fresh assay buffer was added to the receiver chamber. In experiments evaluating the effect of P85 added to the basolateral side of BBMEC, R123 in assay buffer was added to the donor chamber, and 0.1% P85 solution was added to the receiver chamber. At 0-, 15-, 30-, 60-, and 90-min time points, the solutions in the receiver chamber and aliquots (20 µl) from the donor chamber were removed for the determination of the R123 concentration. Fresh assay buffer or 0.1% P85 solution correspondingly was immediately added to the receiver chamber. The amount of R123 in the samples was determined using a Shimadzu RF5000 fluorescent spectrophotometer. All transport experiments were conducted at 37°C and in triplicate.

Fluorescent Microscopy. BBMEC grown on chamber slides (Fisher, St. Louis, MO) were incubated with 0.1% P85-FITC in assay buffer for 2 h at 37°C. After this period, cells were treated for 10 min with a staining solution containing 100 nM mitochondrial dye, MitoTracker-Red (Molecular Probes, Eugene, OR). The loading solution was then removed; cell monolayers were washed three times with ice-cold PBS containing 1% bovine serum albumin and examined using confocal laser microscope ACAS-570, Meridian Instruments (Okemos, MI).

Cytotoxicity Assay. To examine the possible cytotoxic effect of P85, BBMEC were seeded in 96-well plates at a density of 5000 cells/well and allowed to reattach overnight. Then, the cells were exposed to various concentrations of P85 solutions for 2 h at 37°C. After this treatment, cells were washed three times and cultured for three days in the medium. The cytotoxic effects were determined using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay (Ferrari et al., 1990). All experiments were repeated eight times. No cytotoxic effects of P85 were observed when the cells were exposed to P85 for 2 h at up to 5% wt. concentration.

Statistical Analysis. All statistical tests were performed by Microsoft Excel 97 SR-1 program (Microsoft, Redmond, WA) using the two-tailed heteroscedastic t tests. A minimum p value of 0.05 was estimated as the significance level for all tests. S.E.M. values for R123 accumulation levels, microviscosity, and ATP measurements were less than 10%.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Transport of P85 in BBMEC Monolayers

P85 Accumulation in BBMEC Monolayers. Radiolabeled [³H]P85 was used in the experiments examining the interaction of the block copolymer with the BBMEC monolayers. The time- and concentration-dependent accumulation of [³H]P85 in BBMEC was examined. As seen in Fig. 1A, the accumulation kinetics of [³H]P85 (0.0007%) displayed two distinct phases. During the first 30 min of incubation, the amount of the cell-bound [³H]P85 rapidly increased. After 30 min, the amounts of the cell-bound [³H]P85 plateaus in the BBMEC monolayers.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Kinetics (A) and concentration dependence (B) of [3H]P85 accumulation in BBMEC monolayers. Insert in Fig. 1B shows an expanded concentration dependence of accumulation with unimer concentrations of [3H]P85 below 0.012% wt. Concentration of [3H]P85 in kinetics experiment (A) was 0.0007% (0.5 µCi/ml). prot, protein.

Intracellular Localization of P85. The distribution of P85 in BBMEC monolayers was examined using confocal microscopy. For this study, P85 was labeled with FITC, as described previously (Kabanov et al., 1992). Figure 2 presents the confocal fluorescence microphotographs of BBMEC monolayers treated with FITC-P85 (0.1%) for 2 h. In the same experiment BBMEC monolayers were stained with a mitochondrial dye, MitoTracker-Red, to visualize localization of mitochondria. Figure 2A shows the fluorescence of FITC-P85, whereas Fig. 2B shows the fluorescence of MitoTracker-Red dye. These data suggest that the block copolymer is internalized within the cells and is localized primarily in the cytoplasmic compartments. Localization of FITC-P85 in BBMEC monolayers is practically identical to the MitoTracker-Red staining pattern observed in these cells. Therefore, the block copolymer spreads throughout the cell where it may interact with various intracellular targets, including the same organelles where the MitoTracker-Red is accumulated.

View larger version (82K): [in this window] [in a new window]   Fig. 2.   Confocal microscopy showing intracellular localization of FITC-P85 (A) and MitoTracker-Red (B) in BBMEC monolayers. Cells were exposed to 0.1% of FITC-P85 for 2 h, washed with PBS, and stained with 100 nM MitoTracker-Red.

Kinetic Studies of P85 Effects in BBMEC Monolayers

P85 Binding with Cell Membranes. Pluronic block copolymers have a tri-block structure containing central hydrophobic poly(propylene oxide) segment flanked by two hydrophilic poly(ethylene oxide) segments. Hydrophobic poly(propylene oxide) segments can incorporate in the lipid membrane and induce changes in membrane structure (Kostarelos et al., 1999). Interactions of P85 with BBMEC membranes were evaluated in a fluorescence polarization study using DPH as a membrane probe. DPH is a hydrophobic fluorescent compound that spontaneously incorporates in the hydrocarbon regions of the lipid membranes (Laat et al., 1977). Transfer of DPH from the aqueous environment into the cell membranes results in a drastic increase in the intensity of the fluorescence emission of this probe. Furthermore, once the probe is incorporated in the lipid membranes, its fluorescence polarization is strongly dependent on the microenvironment. This provides valuable information concerning membrane structure, specifically, membrane microviscosity. The limitation of this approach, however, is that DPH binds not only with the plasma membranes but also with other membranes within the cells, which can all contribute to the net polarization value measured (Pagano et al., 1977). Consequently, once the polarization changes are observed, it is difficult to discriminate which membranes (i.e., plasma or intracellular organelle) are affected. With this limitation in mind, we examined the time-dependent changes in the fluorescence polarization of DPH in BBMEC following exposure to P85. The microviscosity values were calculated from the polarization measurements as described under Materials and Methods. As seen in Fig. 3A, there was a rapid decrease in the membrane microviscosity following addition of 0.1% P85 to the cell suspension. After 15 min of exposure to P85, the microviscosity was leveled-off and then remained constant throughout the duration of the experiment. This suggests that the P85 molecules rapidly adhered to the cell membranes and incorporated into them, resulting in changes in the structure of the lipid bilayers observed with DPH.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Kinetic effects of P85 on microviscosity (A), intracellular ATP content (B), and R123 accumulation (C) in BBMEC monolayers; cells were treated with 0.1% P85 (filled circles) or assay buffer (open circles) with 3.2 µM R123 (C) at 37°C. prot, protein.

Kinetics of ATP Depletion. Figure 3B presents the time-dependent changes in intracellular ATP levels in BBMEC monolayers during treatment with 0.1% P85. The time dependence observed in this study is similar to that observed in the fluorescence polarization measurements. Specifically, exposure of the cells to the block copolymer resulted in a rapid decrease in intracellular ATP levels within ~15 min of exposure. After 15 min of exposure, the ATP levels remained constant (Fig. 3B).

Time Dependence of Pgp Inhibition. Measurement of the cellular accumulation of R123, a substrate of Pgp, has been commonly used for evaluation of the functional activity of this efflux system in cells (Jancis et al., 1993; Lee et al., 1994, Fontaine et al., 1996). Therefore, to evaluate the time dependence of P85 effect on Pgp efflux, we examined the kinetics of R123 accumulation in BBMEC monolayers exposed to either 0.1% P85 solution or block copolymer-free assay buffer. As seen in Fig. 3C, there was no difference in the R123 levels in BBMEC monolayers in P85-treated and control groups during the first 15 min of incubation. However, at approximately 30 min and continually throughout the duration of the experiment, P85 induced significant increases in the R123 levels compared with those in the control monolayers. This suggests that there is a lag period in the inhibition of Pgp efflux system by P85 in BBMEC monolayers. This period appears to be somewhat higher than the periods (~15 min) needed for leveling off of the microviscosity and intracellular ATP levels as discussed above. However, due to the nature of accumulation experiments, one should expect some delay between actual Pgp inhibition and exhibition of significant differences in R123 intracellular levels.

Dose-Dependent Effects of P85 in BBMEC Monolayers

Effects of P85 on Intracellular ATP. To examine effects of P85 on cellular energy metabolism, the intracellular ATP content was measured using luciferin-luciferase assay (Garewal et al., 1986). Confluent BBMEC monolayers were exposed to various concentrations of P85 in the assay buffer for 2 h. As seen in Fig. 4A, treatment of BBMEC with P85 solutions (0.01% wt. and higher) caused a dramatic decrease in the intracellular ATP levels. To assure that the changes in the ATP levels were not due to increased efflux of ATP out of the cells, the ATP content in the extracellular media was determined following exposure of the cells to P85. Figure 4A shows that there was practically no leakage of ATP into the surrounding media. Therefore, P85 affects energy metabolism in BBMEC, significantly reducing the amount of intracellular ATP.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of P85 on intracellular ATP (filled circles) and ATP in extracellular media (open circles) (A) and R123 accumulation in BBMEC monolayers (B). Cells were treated with various doses of P85 alone (A) or with 3.2 µM R123 in P85 various concentration solutions (B), and aliquots from equal volumes of extracellular media or cell lysates were collected for ATP (A) or R123 (B) quantification. prot, protein.

Effects of P85 on R123 Accumulation. Miller et al., 1997 were first to report the concentration-dependent effects of P85 on R123 accumulation in BBMEC monolayers. In this study, we repeated the earlier experiment to compare the dose dependence of P85 effects on the ATP levels and the Pgp efflux systems. As seen in Fig. 4B, exposure of the cells to the low concentrations of P85 (from ~0.001 to 0.01%) resulted in increased R123 accumulation, consistent with the inhibition of the Pgp efflux system in the BBMEC monolayers. Maximal R123 accumulation was observed at 0.01% P85, which is close to the CMC of this block copolymer. This result is consistent with the earlier reports suggesting that the unimers of Pluronic block copolymers are responsible for the inhibition of Pgp in the cells (Miller et al., 1997; Batrakova et al., 1998a,b, 1999a; Evers et al., 2000). At higher concentrations of P85 (0.1-5%), the R123 levels decrease. The effect of high P85 concentrations is believed to be due to incorporation of the probe in the P85 micelles resulting in the decrease in the amounts of the free probe available for diffusion into the cells (Batrakova et al., 1998b).

Effect of P85 on Pgp ATPase Activity. The effects of P85 on the Pgp ATPase activity were evaluated using membranes containing human Pgp. In this experiment, the membranes were exposed to various treatment solutions, and then the ATPase activity of Pgp was assayed by determining the liberated inorganic phosphate (Druekes et al., 1995). The treatment solutions included the copolymer-free buffer control and solutions containing various concentrations of P85. Additional treatment groups in which the Pgp substrate, verapamil, was added to either copolymer-free buffer or P85 solutions was also evaluated. The purpose of including the verapamil groups was to determine whether binding of a specific substrate with Pgp could modulate the effects of P85 on Pgp ATPase activity. As seen in Fig. 5, P85 induced dramatic decreases in Pgp ATPase activity compared with the copolymer-free control. This inhibitory effect was observed at concentrations of P85 as low as 0.001% as well as at the higher concentrations examined (up to 1%). Furthermore, the inhibitory effect of P85 was observed in the presence of verapamil at all block copolymer concentrations examined (0.001-1%). Verapamil alone in the absence of the block copolymer induced a significant increase in Pgp ATPase activity. This effect is believed to be due to the binding of verapamil in the active center of Pgp (Rebbeor and Senior, 1998; Shepard et al., 1998). Furthermore, it is noteworthy that both in the presence and absence of verapamil, the Pgp ATPase activity was in part restored at 1% P85. In these treatment groups, the Pgp ATPase activity reached about 40 to 45% of that observed in the verapamil- and P85-free controls.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of P85 alone (filled bars) and P85 in the presence of 20 µM verapamil (empty bars) on Pgp ATPase activity in human MDR1 membranes. prot, protein.

Elucidation of Critical Factors in Pgp Inhibition

Effect of the Energy Supplementation on Pgp Efflux in the Presence of P85. To evaluate the relationship between P85-induced changes in ATP levels and the function of Pgp efflux system, the effect of P85 on intracellular ATP content and R123 accumulation was examined in BBMEC monolayers. The following study evaluated whether energy supplementation could restore Pgp efflux function in BBMEC in the presence of P85. The BBMEC monolayers were exposed for 2 h to R123 alone or R123 formulated with 0.1% P85. In an attempt to bypass P85-induced energy depletion, an additional treatment group was also included in this study, in which R123/P85 was supplemented with 50 µM ATP and 10⁵ M dodecylamine, as a permeabilizing agent. As previously reported (Slepnev et al., 1992), treatment of the cells with dodecylamine in combination with P85 allows transport of ATP into the cells from the extracellular media. As seen in Fig. 6, there was an inverse correlation between the R123 uptake and ATP intracellular levels. First, in the presence of 0.1% P85, the ATP level was decreased whereas the probe accumulation was increased compared with the assay buffer controls. Second, when the P85 treatment was combined with the use of an energy supplementation system that elevated intracellular ATP levels, R123 uptake was drastically decreased. This indicates that the function of Pgp was restored. Exposure of BBMEC monolayers to 0.1% P85 and 10⁵ M dodecylamine alone without extracellular ATP neither increased ATP intracellular level, nor decreased R123 accumulation (data not shown). This provides strong evidence supporting the relationship between the ability of P85 to decrease ATP levels and inhibit the Pgp efflux system in BBMEC monolayers.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Relationship between ATP intracellular content (empty bars) and R123 accumulation (filled bars) in BBMEC monolayers. Along with control (first group) and 0.1% P85 solution (second group), cells were treated with energy supplementation system: 0.1% P85, 50 µM ATP and 105 M dodecylamine (DA, third group). prot, protein.

Removal of P85 from Cell Monolayers. In the following studies, BBMEC monolayers were first exposed to P85 for 2 h and then, in the attempt to remove the block copolymer, the cells were incubated with copolymer-free assay buffer for various time intervals. The experiment was first carried out using a suspension of BBMEC labeled with DPH as described earlier to determine the microviscosity of the cell membranes. As seen in Fig. 7A, after P85 solution was removed, the microviscosity of cellular membranes was rapidly increased from about 1.2 to 2.4 poise during first 60 min. After this period, the restoration of microviscosity slowed down with complete restoration of membrane microviscosity (to the level of 3.3 poise determined prior to the exposure of the cells to P85) occurring only 30 to 35 h following the removal of P85 (Fig. 7B). This indicates that the clearance of P85 in BBMEC is a two-phase process. It is postulated that within the first hour, the molecules incorporated into the plasma membrane were washed out. After that, the slower recovery phase represents removal of P85 molecules associated with the intracellular membranes.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Changes in the microviscosity (filled circles), R123 accumulations (empty circles) and intracellular ATP content (empty squares) in BBMEC monolayers following P85 removal. A, short-term changes (up to 140 min); B, long-term changes (up to 100 h). Cells were exposed to 0.1% P85 solution for 2 h, then P85 was washed out, and cells were maintained in copolymer-free media for various periods of time. After that, microviscosity, R123 accumulation, and ATP intracellular level were determined. The dashed lines show the R123 accumulation level in control cell monolayers treated with R123 in assay buffer (A) and the microviscosity and ATP intracellular level in nontreated control BBMEC (B). prot, protein.

Directionality of P85 Effects in BBMEC Monolayers. Previous studies using polarized monolayers of Pgp-expressing human colon epithelial cells, Caco-2, suggested that the effects of P85 on the drug efflux system were direction-dependent (Batrakova et al., 1998b). In these studies, the drug efflux system appeared to be inhibited only when P85 was added at the AP side of the monolayers where Pgp was localized. No inhibition of the drug efflux system was observed when the block copolymer was added at the BL side of the monolayers. Since the BBMEC monolayers have similar directionality of the efflux system (i.e., Pgp is expressed at the AP but not on the BL side), the Caco-2 study raises the question of whether the effects of P85 in BBMEC monolayers are also direction-dependent. Furthermore, in view of the apparent relationship between the Pgp function and energy depletion in BBMEC monolayers, it appeared important to examine whether the energy depletion induced by P85 is dependent on which side of the monolayers, AP or BL, the block copolymer is applied.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Directionality effects of P85 on ATP intracellular level (A) and AP to BL flux of R123 in BBMEC monolayers (B). Cell monolayers were treated with 0.1% P85 from AP side (filled circles) or BL side (empty circles). One group of cell monolayers was pretreated for 30 min with 0.1% P85 added at BL side and then, during R123 flux study, treated with 0.1% P85 added at BL side (empty diamonds). Control monolayers were treated with R123 in assay buffer (filled triangles). prot, protein.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The objective of the current study was to evaluate the mechanism by which P85 inhibits the Pgp efflux system in brain microvessel endothelial cells. Two potential routes by which P85 could inhibit Pgp activity in BBMEC monolayers were evaluated: 1) effect of P85 on the energy conservation and 2) effect of P85 on membrane fluidization. The basis for examining the effects of P85 on ATP was the earlier reports that Pluronic block copolymers could influence mitochondria function and energy conservation in cells. It has long been known that nonionic polymeric detergents, such as Tween 80 and Pluronic, can decrease oxidative metabolism of tissue, cells, and isolated mitochondria (Kirillova et al., 1993). Slepnev et al. (1992) were the first to demonstrate that intracellular levels of ATP were depleted following a 2-h exposure of Jurkat T-cell lymphoma cells to P85. The importance of intracellular ATP in the effects of P85 on Pgp activity is suggested by the recent study (Batrakova et al., 2000). In these studies, the effects of the P85 on ATP levels in several cell types that either do or do not express Pgp were compared. Exposure of resistant and sensitive cells to different doses of P85 resulted in a transient energy depletion that was reversed following removal of the block copolymer. However, cells that expressed Pgp were much more responsive to P85, exhibiting profound decreases in ATP levels at substantially lower concentrations of the block copolymer compared with the sensitive cells. Although the reasons for the elevated responsiveness in the Pgp-expressing cells to the block copolymers still remain unknown and are under investigation in our laboratory, the energy depletion induced by the block copolymers could contribute to their activities in the Pgp-expressing cells.

In the present study, the effects of P85 on the energy pool available in BBMEC monolayers that are known to overexpress drug efflux transporters were examined. This study demonstrates that exposure to P85 induces a dramatic decrease in ATP levels in BBMEC monolayers. This energy depletion by P85 was not due to leakage of intracellular ATP out of the cell, because no ATP was detected in the external media. Therefore, ATP depletion was likely to be a result of inhibition of cellular metabolism rather than due to a loss of ATP in the environment.

The mitochondria are responsible for carrying out much of the metabolic activities of the cell and might be a potential site of action for P85. The likely components contributing to the antimetabolic effects of nonionic surfactants include their ability to serve as K⁺ ionophores (Brierley et al., 1972) and uncouple oxidative phosphorylation (Brustovetskii et al., 1991; Rapoport et al., 2000). It is also possible that these surfactants directly inhibit NADH dehydrogenase by interacting with the hydrophobic sites of this complex in the mitochondrial membrane (Brierley et al., 1972; Kirillova et al., 1993). A recent study by Rapoport et al. (2000), using lipophilic spin-probes, has directly shown that two Pluronic copolymers, P85 and P105, reduce the activity of the electron transport chains in mitochondria as assessed by the rates of bioreduction of these probes in HL-60 cancer cells. The finding that Pluronic block copolymers inhibit respiration in living cells indicates that these molecules are transported inside the cells and reach the mitochondria (Brierley et al., 1972; Kirillova et al., 1993). This, in fact, was directly shown for P85 in the present study using the confocal microscopy. The microscopy study indicates that the block copolymer is transported inside BBMEC monolayers and spreads throughout the cell, where it may interact with intracellular organelles including mitochondria. The accumulation studies using radioactively labeled P85 indicates that the single chains of the block copolymer are more efficiently taken up in the cells than the P85 chains incorporated within the micelles. This is probably due to the smaller size of the unimers compared with the micelles as well as the ability of unimers to bind with the cell membranes. Despite the increased accumulation of P85 unimers, the present study also suggests that substantial amounts of P85 can be accumulated in BBMEC monolayers exposed to the micelle concentrations of the block copolymer. At micellar concentrations of the block copolymers, cellular accumulation of P85 is likely the combination of both unimer and micellar transport processes.

It is tempting to suggest that energy depletion could be one basic reason for inhibition of ATP-dependent Pgp efflux system in BBMEC monolayers. However, energy depletion may be just one of several biochemical events contributing to the action of Pluronic block copolymers. Using the hydrophobic membrane probe DPH, the present study demonstrated that P85 induces drastic changes in the microviscosity of cell membranes in BBMEC. Similar changes in membrane microviscosity were previously observed in the cancer cells treated by Pluronic block copolymers (Melik-Nubarov et al., 1999). These changes can be attributed to the alterations in the structure of the lipid bilayers because of adsorption of the block copolymer molecules on the membranes. Membrane fluidization by various agents including nonionic surfactants, such as Tween 20, Nonidet P-40, and Triton X-100, is known to contribute to inhibition of Pgp efflux function (Regev et al., 1999). Based on the current studies, it is proposed that membrane fluidizers abolish Pgp ATPase activity resulting in the loss of Pgp-mediated drug efflux. This is supported by the observation that P85 inhibits Pgp ATPase activity, and inhibition of this activity is observed with the same doses of the block copolymer as those that inhibit Pgp efflux in BBMEC monolayers.

Therefore, it is likely that these Pluronic block copolymers have a "double-punch" effect in BBMEC monolayers: through ATP depletion and membrane fluidization, which both have a combined result of potent inhibition of Pgp. Involvement of the energy depletion component is demonstrated in the ATP supplementation studies, which suggest that Pgp function in the presence of P85 was restored when ATP levels in BBMEC monolayers were. Since the block copolymer in this experiment was still present and, presumably, bound with the BBMEC membranes, the ATP supplementation study indicates that membrane fluidization alone may not be sufficient for inhibition of the Pgp efflux system in these cells. On the other hand, the directionality studies and experiment involving P85 removal from BBMEC monolayers suggest that energy depletion alone in the absence of interaction of the block copolymer with the Pgp-containing membranes might be insufficient to inhibit the efflux system. Therefore, both factors are critical for exhibition of the effect of P85 on Pgp efflux system in BBMEC monolayers.

The interrelationship between the membrane fluidization and energy depletion components of P85 action can be better understood in view of the current picture of Pgp structure describing Pgp as a two-domain protein with ATP-binding sites in each domain (Ambudkar et al., 1999). Proper interaction of these two ATP-binding sites is crucial for the proper functioning of Pgp. It was suggested that binding of ATP in one domain causes a conformational change in the Pgp molecule necessary for the hydrolysis of ATP and translocation of the substrate (Ramachandra et al., 1998). Therefore, the structural perturbations in the lipid membranes induced by P85 may decrease the affinity of ATP to its binding site and interfere with the ATPase activity. This means that higher concentrations of intracellular ATP would be required for normal functioning of Pgp (i.e., drug efflux system would become more vulnerable to decreases in intracellular ATP). Future studies of the kinetics of the Pgp function in the presence of P85 are needed to confirm this hypothesis.