©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of the P-glycoprotein Multidrug Transporter with Peptides and Ionophores (*)

Frances J. Sharom (§) , Giulio DiDiodato (¶) , Xiaohong Yu , Katherine J. D. Ashbourne (**)

From the (1) Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario N1G 2W1, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

P-glycoprotein functions as an ATP-driven active efflux pump for many cytotoxic drugs. We now show that hydrophobic peptides and ionophores also interact with the multidrug transporter. Multidrug-resistant cells are cross-resistant to several hydrophobic peptides and ionophores, but not to some other membrane-active species. Linear peptides, cyclic peptides, and ionophores stimulated the ATPase activity of P-glycoprotein in plasma membrane vesicles by up to 2.5-fold. Drugs and chemosensitizers were able to block P-glycoprotein ATPase stimulation by verapamil, however, peptides and ionophores (with the exception of cyclosporine A) were unable to do so. Peptides and ionophores also effectively inhibited ATP-dependent drug transport by P-glycoprotein in plasma membrane vesicles. The median effect analysis was used to extract quantitative parameters from the drug transport inhibition data. Unlike drug substrates and cyclic peptides, linear peptides did not inhibit photoaffinity labeling of P-glycoprotein by [H]azidopine. Taken together, these results indicate that certain hydrophobic peptides and ionophores are P-glycoprotein substrates, however, they affect the transporter in a different manner from drugs. Linear peptides interact with P-glycoprotein at a site distinct from those for verapamil and azidopine, whereas the interaction site for cyclic peptides and ionophores appears to be linked to these sites to varying degrees. Export of hydrophobic peptides may be an important physiological function of P-glycoprotein.


INTRODUCTION

The resistance of tumors to multiple chemotherapeutic drugs is a serious barrier to the treatment of human cancer. A common form of multidrug resistance is caused by the overexpression of a 170-180-kDa plasma membrane protein, the P-glycoprotein or multidrug transporter. This protein is a member of the ABC (ATP-binding cassette) (1) or traffic ATPase (2) superfamily, and is proposed to function as an ATP-driven drug efflux pump. Recently, we reconstituted P-glycoprotein into proteoliposomes, and demonstrated that it is indeed an active transporter, pumping drugs up a concentration gradient, powered by concomitant ATP hydrolysis (3) . One intriguing aspect of P-glycoprotein biochemistry concerns its ability to interact with, and transport, many structurally distinct classes of compounds, which suggests the possibility of multiple or overlapping drug-binding domains on the transporter. Drugs transported by P-glycoprotein are, in general, lipophilic, and it has been proposed that the drug-binding site(s) may reside within the transmembrane domains. This hypothesis is consistent with reports that these regions of P-glycoprotein are photolabeled by hydrophobic ligands (4) , and site-directed mutations within the transmembrane segments are able to modulate drug specificity (5, 6, 7) .

Two P-glycoprotein isoforms are expressed in normal tissues: overexpression of Class I and II P-glycoproteins ( e.g. human MDR1, mouse mdr1/3, hamster pgp1/2) leads to multidrug resistance, whereas overexpression of Class III proteins (human MDR3, mouse mdr2, hamster pgp3) does not. Recent studies of mdr2 ``knockout'' mice suggest that the mdr2 gene product plays an essential role in the liver in the export of phospholipid from the apical surface of the canalicular membrane into the bile (8) . This proposal was confirmed by Ruetz and Gros (9) , who demonstrated that the mdr2 protein acts as a phosphatidylcholine translocase (or flippase).

The physiological substrates for Class I and II P-glycoproteins have not yet been identified. However, the sequence similarity of P-glycoprotein with other ABC transporters known to export peptides, both in prokaryotes ( e.g. the Escherichia coli hemolysin exporter HlyB (10) ; the oligopeptide permease of Salmonella typhimurium (11) ) and eukaryotes ( e.g. the yeast ste6 a-factor exporter (12) ; the Tap-1/2 peptide transporters in the endoplasmic reticulum (13) ), suggests that peptides may also serve as P-glycoprotein substrates in vivo. Recently, there have been reports which indicate that P-glycoprotein may transport peptides, although the evidence for this has, to date, been indirect. Sharma et al. showed that mdr1-expressing cells were resistant to the hydrophobic tripeptide ALLN()(14) . In addition, the ability of mdr1 to complement ste6 mutations (15) implies that P-glycoprotein can export the a-factor mating peptide from Saccharomyces cerevisiae, although its efficiency remains uncertain. More recently, we have demonstrated that the channel-forming linear hydrophobic peptide gramicidin D is a substrate for the multidrug transporter, which interferes with the ability of the peptide to form a functional dimeric cation channel in the membrane (16) . In addition, prenylcysteine methyl esters and various hydrophobic peptides have been reported to stimulate the ATPase activity of P-glycoprotein in human mdr1-transfected Sf9 cell membranes (17) and MDR breast cancer cells (18) , which suggests that they interact with the transporter.

Chemosensitizers, compounds which reverse drug resistance, show promise when combined with chemotherapeutic agents in cancer treatment. The identification of new clinically effective chemosensitizers is an important goal in developing strategies to overcome MDR. The cyclic peptide cyclosporine A, a well-known chemosensitizer, was recently reported to be vectorially exported by monolayers of MDR cells (19) and brain endothelial cells (20) , indicating that it is also a substrate for transport by P-glycoprotein. Peptides thus represent an important, and hitherto largely unexamined, class of P-glycoprotein substrates and chemosensitizers, which may offer the hope of substantially lower toxicity.

In the present study, we show that many hydrophobic peptides and ionophores both block drug transport by P-glycoprotein, and stimulate its ATPase activity, in an in vitro membrane vesicle system. In addition, we correlate these data with cross-resistance of MDR cells to peptides, and investigate their ability to inhibit azidopine photoaffinity labeling of P-glycoprotein. The results establish that certain hydrophobic peptides and ionophores are P-glycoprotein substrates, and also indicate that these classes of compounds interact with the transporter at different sites from those associated with other drugs and chemosensitizers. Export of hydrophobic peptides may thus be an important endogenous function for P-glycoprotein, which suggests that further exploration of this class of compounds as potential chemosensitizers is warranted.


MATERIALS AND METHODS

MDR Cell Lines and Plasma Membrane Preparation

The drug-sensitive parent Chinese hamster ovary cell line (AuxB1) and an MDR cell line selected for colchicine resistance (CHC5) (21) , were grown as described previously (22, 23, 24) . Plasma membrane vesicles from AuxB1 and CHC5 were isolated by a method involving cell disruption by nitrogen cavitation followed by centrifugation on a 35% (w/w) sucrose cushion (22) . Plasma membrane vesicles were stored at 70 °C for no longer than 3 months before use.

Cross-resistance of MDR Cells

Cross-resistance of CHC5 cells to peptides and ionophores, and chemosensitization by verapamil, were determined by growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye reduction assay, as described previously (16, 25) . The fold resistance was calculated as the ratio of the ICvalues for CHC5 relative to the AuxB1 parent. The chemosensitization index was calculated as the ratio of the ICfor CHC5 in the presence of 20 µM verapamil, to that in its absence (16, 25) . A value of the chemosensitization index of 1 indicates that resistance was not reversed by verapamil, whereas a value >1 indicates that resistance was reversed.

Protein Determination

The protein content of CHC5 plasma membrane was determined by a microplate adaptation of the Bradford assay (26) , using bovine serum albumin as a standard.

Measurement of ATPase Activity

The ATPase activity of P-glycoprotein in CHC5 plasma membrane vesicles was determined as described previously (3, 24, 27) , by measuring the release of inorganic phosphate from ATP, using a colorimetric method. Samples contained 1-2 µg of CHC5 plasma membrane with 2 mM ATP and 5 mM Mg, which gave maximal ATPase activity. The assay buffer did not contain either Naor K, to avoid contributions to activity from the NaK-ATPase; addition of 1 mM ouabain to the assay did not affect the measured ATPase activity. Membrane vesicles were preincubated with peptides and ionophores for 5 min before initiation of the assay by addition of ATP. Peptides, ionophores, and drugs were added as stock solutions in MeSO, and controls contained the appropriate levels of MeSO (which never exceeded 1%, v/v).

Measurement of Drug Transport

Steady-state [H]colchicine uptake into CHC5 plasma membrane vesicles was determined using a protocol previously developed in our laboratory (23) . Briefly, membrane vesicles (25-35 µg of protein in a 100-µl final volume of buffer) were mixed with 1 µM [H]colchicine (0.3 µCi/sample), 5 mM Mg, and 1 mM ATP plus a regenerating system (creatine phosphate/creatine kinase). After various times at 23 °C, the vesicles were harvested by rapid filtration on Whatman GF/F filters using a Hoeffer filtration manifold, and immediately washed with 5 ml of ice-cold buffer. Filters were dried and radioactivity was quantitated by liquid scintillation counting. Peptides, ionophores, and drugs were added as stock solutions in MeSO, and controls contained the appropriate levels of MeSO (which never exceeded 1%, v/v). Drug binding to filters, and nonspecific uptake into the vesicles, were determined in the absence of membrane vesicles, and in the absence of ATP and a regenerating system, respectively.

Median Effect Analysis

The median effect equation (28, 29) describes the relationship between any concentration of a compound, and its effect on the system being studied. In this case, the inhibition of [H]colchicine transport into CHC5 plasma membrane vesicles was measured at various concentrations of the test compound(s). The basic median effect equation is as follows,

  

On-line formulae not verified for accuracy

Assay for Membrane Permeabilization

The effect of peptides and ionophores on the integrity of lipid bilayers was determined by a modification of the method of Ertel et al. (30) . Briefly, 2.5 mg of egg phosphatidylcholine (Sigma) in ethanol was dried under a stream of Nand then pumped in vacuo for 30 min. The lipid was then suspended in 1 ml of 70 mM calcein (Sigma), 50 mM NaCl. After freeze-thawing 10 times in liquid N, large unilamellar vesicles (LUV) were prepared by high pressure extrusion through 100-nm polycarbonate filters, as described (31) . Untrapped dye was removed by gel filtration chromatography on Sephadex G-50. The void volume fractions containing LUV were pooled, and the lipid content determined using a microscale Bartlett assay (32) . Aliquots (0.5-1.0 µg of lipid) were dispensed into individual wells of a 96-well microplate, in a total volume of 100 µl of Hepes-buffered saline (10 mM Hepes, 0.15 M NaCl, 5 mM EDTA, pH 7.0), and increasing concentrations of the test compounds in Hepes-buffered saline were added. The release of calcein from the LUV was quantitated after 30 min at 23 °C, using fluorescence measurements on a cytofluorimeter (Millipore Cytofluor 2300; = 485 nm, = 535 nm). [H]Azidopine Photoaffinity Labeling-Photoaffinity labeling of P-glycoprotein in CHC5 membrane vesicles with [H]azidopine (200 nM, 52 Ci/mmol; Amersham) was carried out as described previously (16, 25, 33, 34) , in the presence of various concentrations of peptides and ionophores. Membrane vesicles were then analyzed by SDS-polyacrylamide gel electrophoresis on a 7.5% polyacrylamide gel, followed by fluorography.


RESULTS

MDR Cells Are Cross-resistant to Peptides and Ionophores

The MDR Chinese hamster ovary cell lines CHC5 and CHB30 were previously selected for resistance to colchicine, and overexpress predominantly the Class I isoform of P-glycoprotein (35) . We previously reported that CHC5 cells and mdr1-transfectants were highly cross-resistant to the linear channel-forming peptide gramicidin D (16) . We have now investigated cross-resistance of MDR cell lines to a selected set of linear and cyclic hydrophobic peptides and ionophores, and chemosensitization of this resistance by verapamil (). CHC5 displayed high levels of resistance to the protease inhibitor ALLN, and moderate levels were noted for the related peptide ALLM and the cyclic peptide ionophore valinomycin. A low, but significant level of resistance was observed for nonactin, a cyclic non-peptide ionophore. The CHB30 cell line, which was derived from CHC5, has substantially higher levels of drug resistance and P-glycoprotein expression, and displayed 5-fold resistance to nonactin, indicating that cross-resistance to this compound is genuine. No cross-resistance was observed to the membrane-active linear peptides melittin and alamethicin, or to the cyclic membrane-active peptide gramicidin S, and the cyclic peptide chemosensitizer cyclosporine A. Verapamil was able to reverse resistance to ALLM, ALLN, gramicidin D, valinomycin, and nonactin, which suggests that they are all P-glycoprotein substrates, but had no effect on resistance to melittin, alamethicin, gramicidin S, and cyclosporine A ().

P-glycoprotein ATPase Activity Is Stimulated by Drugs and Chemosensitizers

CHC5 plasma membrane vesicles displayed a high intrinsic Mg-ATPase activity (0.273 µmol/min/mg) compared to plasma membrane from the drug-sensitive parent line AuxB1 (0.056 µmol/min/mg, see Ref. 24). In previous studies, we confirmed that this additional ATPase activity in the drug-resistant cell line arises from the presence of P-glycoprotein (24) . The ATPase activity of CHC5 membrane was inhibited by vanadate (ICof 1.5 µM; 80% inhibition at 10 µM), which was previously observed to inhibit both P-glycoprotein-mediated drug transport (23) and the ATPase activity of a partially purified P-glycoprotein preparation (24) .

The ATPase activity of CHC5 plasma membrane was stimulated over 2.2-fold by the chemosensitizer verapamil. Trifluoperazine also produced a large activation, while the drug substrates vinblastine and colchicine increased activity by 40%. Maximal stimulation of P-glycoprotein ATPase was reached at 10 µM for verapamil, trifluoperazine, and vinblastine, and 100 µM for colchicine. The ATPase activity of plasma membrane from the drug-sensitive AuxB1 parent cell line was not stimulated significantly by any of the compounds tested. Thus, P-glycoprotein ATPase in a native membrane environment is activated by certain chemosensitizers and drugs. Similar results have been reported for plasma membrane vesicles from other P-glycoprotein-expressing cells. CHC5 membrane vesicles therefore provide a simple and convenient system for screening various compounds for their effect on P-glycoprotein ATPase activity. We previously demonstrated that the ATPase activity of P-glycoprotein in detergent solution is also modulated by drugs (24, 36) , although to a lesser extent.

Peptides and Ionophores Stimulate P-glycoprotein ATPase Activity

Linear and cyclic peptides and ionophores were examined for their ability to stimulate P-glycoprotein ATPase in CHC5 plasma membrane. The linear peptides ALLN, ALLM, pepstatin A, and leupeptin (Fig. 1 A), and the cyclic ionophores valinomycin and nonactin (Fig. 1 B) produced maximal enhancement of ATPase activity of around 2-2.5-fold. These six compounds all exhibited a pattern of activation which did not decrease at high concentrations, unlike the biphasic pattern noted previously by our group (3, 24, 36) and others (37, 38) for chemosensitizers and drugs. The membrane-active linear peptides melittin, alamethicin, and gramicidin D did not stimulate the ATPase (Fig. 1 A); instead they caused varying degrees of inhibition as the concentration increased. Activity was also stimulated (up to 1.4-fold) by cyclosporine A, which displayed a biphasic pattern, whereas gramicidin S did not activate the ATPase (Fig. 1 B).


Figure 1: Stimulation of P-glycoprotein ATPase activity in CHC5 plasma membrane by peptides and ionophores. CHC5 plasma membrane vesicles (1.5-2.0 µg of protein) were assayed for Mg-ATPase activity in the presence of various linear peptides ( A) ALLM (), ALLN (▾), leupeptin (), pepstatin A (), gramicidin D (), melittin (), alamethicin (); and various cyclic peptides and ionophores ( B) gramicidin S (), valinomycin (), cyclosporine A (), nonactin (▾). Data are presented as percent control ATPase activity (means ± S.E., n = 3), measured in the absence of peptides.



The concentrations of each peptide or ionophore that induced half-maximal stimulation of P-glycoprotein ATPase activity (SCvalues) were interpolated from the curves in Fig. 1, A and B, and are listed in , together with the maximal fold-stimulation observed for each. The SCvalues are useful indicators of the relative ``affinity'' of each compound for interaction with P-glycoprotein. Cyclosporine A showed the lowest SCvalue of all the compounds tested (around 10 nM), whereas the SCvalue for valinomycin was less than 1 µM, comparable to that for vinblastine. Nonactin and pepstatin A gave half-maximal ATPase stimulation in the low micromolar range, while ALLN, ALLM, and leupeptin had SCvalues between 40 and 100 µM. In general, only compounds to which CHC5 cells were cross-resistant stimulated ATPase activity; peptides such as melittin, alamethicin, and gramicidin S, to which MDR cells did not display cross-resistance, did not induce ATPase stimulation.

Drugs Abolish Verapamil Stimulation of P-glycoprotein ATPase, whereas Peptides and Ionophores Do Not

As demonstrated above, verapamil stimulates the ATPase activity of CHC5 plasma membrane by over 2.2-fold. It was of interest to determine whether peptides and ionophores block verapamil-induced ATPase activation, since this would give some indication of whether these classes of compounds compete with verapamil for a common binding site on P-glycoprotein. As shown in Fig. 2, cyclosporine A and vinblastine completely abrogated verapamil stimulation of P-glycoprotein ATPase activity at low concentrations (5 and 50 µM, respectively), as did other drugs and chemosensitizers, including daunomycin, trifluoperazine, quinine, and quinidine (not shown). In contrast, linear peptides such as ALLM and pepstatin A were unable to do so, even at a concentration of 200 µM. Valinomycin inhibited verapamil stimulation slightly at 100 µM. ALLN, leupeptin, and nonactin were also unable to block verapamil stimulation of ATPase activity (not shown). These data support the hypothesis that linear peptides interact with P-glycoprotein at a binding site distinct from that for verapamil. On the other hand, cyclosporine A and other drugs and chemosensitizers reside in a site which either overlaps with the verapamil-binding site, or is allosterically linked to it, so that occupancy affects the ability of verapamil to bind to the transporter, or signal the ATPase catalytic site once bound.


Figure 2: Effect of peptides and ionophores on stimulation of P-glycoprotein ATPase activity by verapamil. CHC5 plasma membrane vesicles (1.5-2.0 µg of protein) were assayed for Mg-ATPase activity in the presence of 10 µM verapamil, and increasing concentrations of ALLM (), pepstatin A ( PEPA, ), valinomycin ( VAL, ▾), cyclosporine A ( CSA, ), and vinblastine ( VBL, ). Data are presented as percent control ATPase activity (means ± S.E., n = 3), measured in the presence of verapamil alone. The basal level of ATPase activity determined in the absence of verapamil is indicated by the horizontal dashed line.



Peptides and Ionophores Inhibit Drug Transport by P-glycoprotein

Peptides and ionophores were examined for their ability to block active, ATP-dependent colchicine transport into CHC5 plasma membrane vesicles, using methodology previously established in our laboratory (3, 23) . As shown in Fig. 3 , a large number of peptides and ionophores inhibited accumulation of drug in the vesicle lumen, in a concentration-dependent manner. Cyclosporine A and vinblastine (Fig. 3 B) were highly effective competitors of colchicine transport, with >90% inhibition observed at 1 and 3 µM, respectively. Valinomycin (Fig. 3 B) also blocked transport very effectively in the low micromolar concentration range. Linear peptides (Fig. 3 A) inhibited colchicine transport in the order of effectiveness pepstatin A > leupeptin > ALLM, and the cyclic ionophore nonactin was the least potent inhibitor. Gramicidin D could not be tested above 50 µM due to poor solubility, but it blocked drug accumulation by >35% at this concentration.


Figure 3: Inhibition by peptides and ionophores of P-glycoprotein-mediated drug transport. Equilibrium uptake of 1 µM [H]colchicine into CHC5 membrane vesicles was measured in the presence of 1 mM ATP and a regenerating system, together with increasing concentrations of ( A) nonactin ( NON, ▾), ALLM (), leupeptin ( LEU, ), pepstatin A ( PEPA, ); and ( B) valinomycin ( VAL, ), vinblastine ( VBL, ), and cyclosporine A ( CSA, ). Data are presented as percent control drug uptake relative to membrane vesicles in the absence of peptides, and represent the mean ± S.E. ( n = 3).



Median Effect Analysis of Transport Inhibition Data

The transport inhibition data presented in Fig. 3 cannot be analyzed by enzyme kinetic models, since it represents steady-state drug accumulation, rather than initial rates of transport. As an alternative means for evaluation of the transport inhibition data, we employed the median effect analysis, which was developed by Chou (for reviews, see Refs. 28 and 29). This method is derived from the law of mass action, and requires no assumptions about mechanism (indeed, it is mechanism-independent), or estimates of binding or kinetic constants. It permits the experimenter to quantitate the relationship between the concentration of any compound, D, and its effect on the system being studied (see ``Materials and Methods''). In this case, the fractional inhibition of colchicine accumulation in CHC5 plasma membrane vesicles (data shown in Fig. 3) was used as the parameter f, and median effect plots of log ( f/ f) versus log D were constructed for each of the test compounds. The drug accumulation inhibition data gave a series of straight line median effect plots (shown in Fig. 4), covering a wide range of concentrations. The plot for cyclosporine A (the most effective transport inhibitor) appears at the far left of the series, and the plot for nonactin (the least effective transport inhibitor) is shown at the far right. The parameter Dwas determined for each plot from the zero intercept on the x axis, and represents the concentration of test compound required to inhibit drug accumulation by 50% (see ). The Dvalues thus provide a means of quantitating the efficacy of each test compound as an inhibitor of drug transport by P-glycoprotein. All of the peptides that stimulated ATPase activity also inhibited colchicine transport, which indicates that they are likely to be P-glycoprotein transport substrates. The linear peptide pepstatin A and the cyclic peptide ionophore valinomycin are comparable to, or more effective than verapamil as inhibitors of the multidrug transporter.


Figure 4: Median effect analysis of transport inhibition data. Median effect plots of log ( f/ f) versus log D for nonactin (▾), ALLM (), leupeptin (), pepstatin A (), valinomycin (), cyclosporine A (), and vinblastine ().



The slope of the median effect plot for each test compound yields the parameter m, which is an indicator of the sigmoidal nature of the plot, analogous to a Hill coefficient. Compounds with m values approximating 1 (non-sigmoidal) include ALLM, leupeptin, pepstatin A, nonactin, and vinblastine. Three peptides (ALLN, valinomycin, and cyclosporine A) and verapamil showed m values close to 3, which indicates a high level of sigmoidicity in the median effect plot.

Membrane Permeabilization by Peptides

Three of the peptides tested (melittin, alamethicin, and gramicidin S) were nonspecific membrane-active agents, and displayed anomalous effects on drug transport by P-glycoprotein. As shown in Fig. 5 A, increasing concentrations of these peptides reduced colchicine accumulation below the level of the control with no ATP. We have previously demonstrated that colchicine uptake into CHC5 plasma membrane vesicles in the absence of ATP represents diffusional equilibration of the drug across the bilayer into the vesicle lumen (23) . Thus, it seems likely that these three peptides nonspecifically permeabilize the membrane, and prevent sequestration of drug in the vesicle lumen by diffusional equilibration. This premise was confirmed by the use of an assay which monitored release of the self-quenching fluorescent dye, calcein, from the lumen of large unilamellar phospholipid vesicles. As shown in Fig. 5 B, melittin and alamethicin released calcein from LUV over the concentration ranges 0.017-0.17 and 0.5-5 µM, respectively, and gramicidin S permeabilized the vesicles at around 3 µM (not shown). The permeabilization curve for the nonionic detergent Triton X-100 is displayed for comparison; it released calcein from the vesicle lumen at about 0.01% (v/v). It was also necessary to address the issue of whether any of the other compounds tested in this study inhibited transport as a result of nonspecific permeabilization, rather than an effect on P-glycoprotein. Further experiments showed that none of the other peptides, ionophores, drugs, or chemosensitizers listed in were able to release calcein from the vesicle lumen within the relevant concentrations ranges; typical data resembled those for leupeptin (Fig. 5 B). Taken together, these observations indicate that melittin, alamethicin, and gramicidin S inhibited drug transport by nonspecific permeabilization of the CHC5 membrane vesicles, whereas the other compounds tested did so as a result of an effect on P-glycoprotein. These results are consistent with other data indicating that MDR cells are not cross-resistant to these three peptides, and that they do not stimulate P-glycoprotein ATPase activity ().


Figure 5: Drug transport inhibition and membrane permeabilization. A, inhibition of P-glycoprotein-mediated drug transport. Equilibrium uptake of 1 µM [H]colchicine into CHC5 membrane vesicles was measured in the presence of 1 mM ATP and a regenerating system, together with increasing concentrations of alamethicin ( ALA, ), gramicidin S ( GMS, ), and melittin ( MEL, ▾). Data points are given as percent control drug uptake relative to membrane vesicles in the absence of added peptides, and represent the mean ± S.E. ( n = 3). B, permeabilization of LUV by various peptides. LUV of egg phosphatidylcholine preloaded with the fluorescent dye calcein were incubated with increasing concentrations of various peptides, including leupeptin ( LEU, ), alamethicin ( ALA, ), and melittin ( MEL, ▾). After 30 min at 23 °C, the release of calcein from the lumen of the vesicles was monitored (= 485 nm, = 535 nm). Results for the nonionic detergent Triton X-100 ( TRIT, ) are shown for comparison.



Effect of Peptides and Ionophores on Azidopine Photoaffinity Labeling of P-glycoprotein

The ability to inhibit photoaffinity labeling of P-glycoprotein by the drug azidopine has frequently been used as an indicator of whether a particular compound is a P-glycoprotein ``substrate'' (33, 34) . In particular, it is believed that compounds which compete with azidopine for a common binding site on the multidrug transporter will be able to block photolabeling. For example, the drug vinblastine abolishes azidopine photolabeling of P-glycoprotein very effectively, with half-maximal inhibition at 5 µM. In contrast, linear peptides were completely unable to block azidopine photolabeling of P-glycoprotein (see ), as shown for ALLN and pepstatin A in Fig. 6 , A and B, even at concentrations comparable to, or higher than, those inhibiting drug transport. This observation suggests that the binding site on P-glycoprotein for linear peptides is distinct from that for azidopine. Alamethicin displayed inhibition of photolabeling, possibly as a result of nonspecific membrane disruption, at concentrations substantially higher than those inhibiting transport. We previously noted that certain amphiphiles, including Triton X-100 and Nonidet P-40, were able to block azidopine labeling of P-glycoprotein in CHC5 plasma membrane (25) .


Figure 6: Effect of peptides and ionophores on photoaffinity labeling of P-glycoprotein by [H]azidopine. CHC5 membrane vesicles (20 µg of protein) were incubated with [H]azidopine in the presence of increasing concentrations of: A, ALLN; B, pepstatin A; C, valinomycin; and D, nonactin. After 1 h at room temperature, the samples were subsequently irradiated with UV light for 30 min. After separation by SDS-polyacrylamide gel electrophoresis, the intensity of P-glycoprotein photolabeling was detected by fluorography; the only visible band was P-glycoprotein, with a molecular mass of 170-180 kDa. Peptide/ionophore concentrations in µM are indicated along the bottom of each gel. Arrows and numbers to the left of the gels indicate the position of molecular mass markers in kDa.



Of the cyclic peptides and ionophores tested, cyclosporine A, valinomycin, and nonactin inhibited photolabeling (Fig. 6, C and D, and ), whereas gramicidin S, which does not appear to be a P-glycoprotein substrate, did not. Valinomycin and cyclosporine A abolished photolabeling at half-maximal concentrations which were of the same order of magnitude as those required for half-maximal inhibition of colchicine transport (see ). It thus seems likely that the site of interaction of these cyclic structures within P-glycoprotein either overlaps with the azidopine site, or is negatively allosterically linked to it.


DISCUSSION

Previous reports in the literature have suggested that P-glycoprotein may transport hydrophobic peptides and ionophores, including ALLN (14) and gramicidin D (16) . The present study establishes that various hydrophobic peptides and ionophores (both linear and cyclic) are P-glycoprotein substrates. Criteria used to classify a particular compound in this way include cross-resistance in MDR cells, and the ability to both stimulate P-glycoprotein ATPase activity, and inhibit colchicine transport by P-glycoprotein in a CHC5 plasma membrane vesicle system. This vesicle model system is unique in that it permits concurrent quantitation of both the latter parameters. We conclude that the CHC5 plasma membrane vesicle system will prove both useful and convenient for rapid screening of putative P-glycoprotein substrates.

We have used the median effect analysis to derive the quantitative parameters Dand m from the equilibrium transport inhibition data gathered in this study. Several previous reports in the literature have mistakenly applied Michaelis-Menten kinetic analysis to this type of data, which is clearly incorrect, since initial rates of transport were not measured. Experiments in our laboratory() have shown that colchicine transport by P-glycoprotein in the CHC5 membrane vesicle system is too fast (on the subsecond time scale) for rigorous measurement of true initial rate kinetics using conventional rapid filtration methodology. Specialized instrumentation for rapid kinetic measurements will be necessary to determine true initial rates of drug transport.

The equilibrium inhibition data for all the compounds examined fitted well to the median effect equation, as indicated by the straight line plots in Fig. 4. The resulting Dvalues quantitate the ability of each compound to block P-glycoprotein drug transport, and are useful indicators of the relative affinity of the interaction of each with the transporter. Values for m, which is a parameter analogous to a Hill number, were also determined for each species. All the compounds tested had m values close to either 1 (not sigmoidal) or 3 (highly sigmoidal). The significance of the value of m with respect to P-glycoprotein function is not yet clear, although it must reflect the underlying molecular interaction. The median effect analysis therefore appears to be a very useful method for quantitatively analyzing equilibrium drug uptake data, which have been reported for several different vesicle systems containing P-glycoprotein (3, 23, 40, 41, 42) .

Any investigation of the effect of various compounds on transport in a vesicle system must consider the possibility of nonspecific permeabilization. This is especially important for studies involving P-glycoprotein, where many putative substrates and chemosensitizers are amphiphilic and/or membrane-active. Three of the peptides in this study (gramicidin S, melittin, and alamethicin) were not P-glycoprotein substrates based on the criteria of cross-resistance and ATPase stimulation, yet they inhibited drug transport. They were shown to permeabilize the CHC5 plasma membrane vesicles in the same concentration range, and thus we concluded that they affected transport in a nonspecific fashion, rather than by an interaction with P-glycoprotein itself. These results indicate that caution is necessary when dealing with membrane-active compounds in transport experiments which depend on membrane integrity.

The other parameter used to identify a compound as a P-glycoprotein substrate is stimulation of ATPase activity. Assuming that the ATPase activity originating from other membrane ATPases is the same in CHC5 and the parent cell line, then approximately 80% of the measured activity in CHC5 membrane can be attributed to P-glycoprotein. Thus, the actual stimulation of P-glycoprotein ATPase activity by verapamil is likely around 2.8-fold. This is similar to the 3.5-fold stimulation observed in plasma membrane from Sf9 insect cells overexpressing the human mdr1 gene product (37) , and lower than the 5-fold stimulation noted for plasma membrane from a Chinese hamster ovary cell line selected for a high level of P-glycoprotein overexpression (39) . Different levels of ATPase stimulation by other drugs were also reported in these two studies. It is possible that these variations arise from differences in the properties of the gene products themselves (human versus hamster), or differences in post-translational modification of P-glycoprotein, especially phosphorylation, which is known to modify drug resistance in intact cells. We recently investigated the phosphorylation state of P-glycoprotein in CHC5 plasma membrane, and determined that it is not fully phosphorylated (36) .

There appears to be little correlation between the turnover rate of ATP hydrolysis following stimulation by peptides and ionophores, and their affinity for P-glycoprotein, as assessed by their ability to compete with colchicine for transport in plasma membrane vesicles. Some compounds, especially the linear tripeptides ALLN, ALLM and leupeptin, and the cyclic ionophore nonactin, induced the highest levels of ATPase activity of all the species tested, yet showed high Dvalues for inhibition of transport, indicating that they interact with P-glycoprotein with relatively low affinity. In contrast, some drug substrates, such as vinblastine, stimulated smaller increases in P-glycoprotein ATPase activity, yet were clearly very high affinity transport substrates on the basis of their Dvalues. However, the concentration of compound required for half-maximal ATPase stimulation (SC) correlated well with the Dvalues for transport inhibition for all the linear peptides tested. In the case of the cyclic peptides and ionophores, ATPase stimulation occurred at concentrations substantially lower (6-70-fold) than those observed to inhibit drug transport.

The results of verapamil blocking and azidopine photoaffinity labeling experiments allow us to come to some conclusions about the relationship between the interaction sites for these drugs and those for the linear and cyclic peptides. Although drugs such as vinblastine and trifluoperazine are able to block ATPase activation by verapamil in a concentration-dependent manner, all of the linear peptides tested were unable to do so. This finding indicates that the P-glycoprotein interaction site for the linear peptides does not overlap with, and is not linked to, the site where verapamil binds. Linear peptides were also incapable of blocking azidopine photolabeling of P-glycoprotein, which suggests that the binding site on the transporter for linear peptides is also distinct from that for azidopine.

Of the cyclic structures tested, the only one capable of abrogating verapamil stimulation was cyclosporine A, which was highly effective at relatively low concentrations. In contrast, cyclosporine A, valinomycin, and nonactin all inhibited photolabeling at concentrations similar to those which blocked drug transport, which suggests that the site of interaction of these cyclic peptides/ionophores within P-glycoprotein either overlaps with the azidopine site, or is linked to it in a negative allosteric fashion. Thus the interaction site on P-glycoprotein for cyclic peptides/ionophores appears to overlap with, or be linked to, the verapamil and azidopine sites to varying degrees. It is also clear from this study that not all P-glycoprotein substrates can be identified on the basis of their ability to block azidopine photolabeling.

The peptide ionophore gramicidin D deserves a special mention. Several studies have shown that it is undoubtedly a P-glycoprotein substrate ( e.g. Refs. 16 and 43), and thus it might be expected to inhibit drug transport and stimulate P-glycoprotein ATPase activity. However, its extremely low aqueous solubility is clearly a problem at the experimental level; we demonstrated only partial inhibition of drug transport (Fig. 3 A), and ATPase stimulation was not observed.

After completion of this work, Sarkadi et al. (17) reported that the ATPase activity of the human MDR1 protein overexpressed in Sf9 insect cell membranes was stimulated by various bioactive hydrophobic peptides. In general, for those peptides tested in both our study and theirs, comparable results were obtained. However, leupeptin was ineffective at P-glycoprotein ATPase stimulation in their system, whereas we have clearly shown that it is a P-glycoprotein substrate on the basis of both ATPase stimulation and inhibition of drug transport. It is possible that the insect cell membrane system contains surface proteases which bind or degrade certain peptides. In addition, the cytotoxic pentapeptide dolastatin 10, a promising new anti-tumor agent, appears to be a P-glycoprotein substrate (44) . Various prenylated cysteine compounds, which are quite hydrophobic, also stimulate P-glycoprotein ATPase activity in the Sf9 system (18) . In both the latter cases, some inhibition of [H]azidopine photolabeling was observed, although at concentrations much higher than those needed to stimulate ATPase activity.

We suggest that the ability of a compound to inhibit drug transport in the CHC5 membrane vesicle system in vitro is a much more reliable indicator of whether it is a P-glycoprotein substrate than the ability of the compound to either stimulate P-glycoprotein ATPase activity, or block azidopine photoaffinity labeling, both of which have been proposed as possible one-step ``screens'' for putative substrates. In addition, the median effect analysis allows quantitation of the inhibition process.

The relatively low Dvalues measured for several of the hydrophobic peptides and ionophores tested in this study, in many cases comparable to those for drugs and chemosensitizers, indicates that this class of compounds interacts with P-glycoprotein with high affinity. These results strengthen the argument that export of hydrophobic peptides may be the true physiological function of the multidrug transporter. Many of the compounds tested in this study are rich in leucine and/or valine, and it is possible that this is one structural characteristic of peptides that is recognized by P-glycoprotein. We are currently examining the interaction of a series of leucine-rich synthetic hydrophobic peptides with the multidrug transporter, in an effort to determine how the chain length, charge, and amino acid R-group affect their interaction with the protein. Preliminary experiments using I-labeled peptides indicate that they are, in fact, transported by P-glycoprotein. Further investigation of the interaction of P-glycoprotein with hydrophobic peptides will provide information necessary for the development and design of new peptide-based chemosensitizers for use in clinical treatment.

  
Table: Effect of peptides and ionophores on various aspects of P-glycoprotein function



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of research grants from the Cancer Research Society (Canada), and the National Cancer Institute of Canada, with funds provided by the Canadian Cancer Society. To whom correspondence should be addressed: Guelph-Waterloo Centre for Graduate Work in Chemistry, Dept. of Chemistry and Biochemistry, University of Guelph, Guelph, ON, Canada, N1G 2W1. Tel.: 519-824-4120 (ext. 2247); Fax: 519-766-1499.

Recipient of a Natural Sciences and Engineering Research Council of Canada post-graduate scholarship.

**
Awarded a Natural Sciences and Engineering Research Council of Canada Undergraduate Student Research Award.

The abbreviations used are: ALLN, N-acetyl-leucyl-leucyl-norleucinal; ALLM, N-acetyl-leucyl-leucyl-methioninal; LUV, large unilamellar vesicles; MDR, multidrug-resistant.

G. DiDiodato and F. J. Sharom, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Victor Ling, Ontario Cancer Institute, for providing the MDR cell lines used in this study.


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