Detergents as intrinsic P-glycoprotein substrates and inhibitors

We assessed the interaction of three electrically neutral detergents (Triton X-100, C₁₂EO₈, and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid-water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, ΔG_tl⁰, that reflects the direct detergent-transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergent-transporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately ΔG_EO⁰ = − 1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.     

P-Glycoprotein kinetics measured in plasma membrane vesicles and living cells

P-glycoprotein (MDR1, ABCB1) is an ATP-dependent efflux transporter of a large variety of compounds. To understand P-glycoprotein in more detail, it is important to elucidate its activity in the cellular ensemble as well as in plasma membrane vesicles (under conditions where other ATP dependent proteins are blocked). We measured P-glycoprotein activity in inside-out vesicles formed from plasma membranes of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) for comparison with previous measurements of P-glycoprotein activity in living NIH-MDR1-G185 cells. In plasma membrane vesicles activity was measured by monitoring phosphate release upon ATP hydrolysis and in living cells by monitoring the extracellular acidification rate upon ATP synthesis via glycolysis. P-glycoprotein was stimulated as a function of the concentration with 19 structurally different drugs, including local anesthetics, cyclic peptides, and cytotoxic drugs. The concentrations of half-maximum P-glycoprotein activation, K1, were identical in inside-out plasma membrane vesicles and in living cells and covered a broad range of concentrations (K1 approximately (10(-8)-10(-3)) M). The influence of the pH, drug association, and vesicle aggregation on the concentration of half-maximum P-glycoprotein activation was investigated. The turnover numbers in plasma membrane vesicles and in living cells were also approximately identical if the latter were measured in the presence of pyruvate. However, in the absence of pyruvate they were higher in living cells. The rate of ATP hydrolysis/ATP synthesis decreased exponentially with decreasing free energy of drug binding from water to the transporter, DeltaG0(tw)(1) (or increasing binding affinity). This suggests that drug release from the transmembrane domains has to occur before ATP is hydrolyzed for resetting the transporter.     

ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells

ATPase activity of multidrug-resistance protein (P-glycoprotein, Pgp) from Chinese hamster ovary cells was studied. Catalytic characteristics were established for Pgp both in its natural plasma membrane environment and in purified reconstituted protein. Generally the two preparations of Pgp behaved similarly, and demonstrated low affinity for MgATP, low nucleotide specificity, preference for Mg-nucleotide, and pH optimum near 7.5. A high-affinity binding site involved in catalysis was not apparent. Effective covalent inactivators were NBD-C1, NEM, 8-azido-ATP, and 2-azido-ATP. DCCD, FITC, and pyridoxal phosphate were only weakly inhibitory. Lipid composition was found to affect the degree of drug stimulation of ATPase in purified reconstituted Pgp, suggesting that the lipid environment affects coupling between drug-binding and catalytic sites, and that Pgp expressed in different tissues could show different functional characteristics.     

P-glycoprotein senses its substrates and the lateral membrane packing density: consequences for the catalytic cycle

P-glycoprotein (ABCB1) prevents absorption (e.g., blood-brain barrier) or enhances excretion (e.g., kidney) by moving substrates from the cytosolic to the extracellular membrane leaflet at the expense of ATP hydrolysis. It translocates various drugs and functions in membranes exhibiting different lateral packing densities. To gain more functional insight, we measured the temperature dependence of the P-glycoprotein ATPase activity in NIH-MDR1-G185 cell membranes in the absence and presence of three drugs (promazine, verapamil, and PSC833), exhibiting significantly different transporter affinities. Activation enthalpies (Delta H(++)) and entropies ( TDelta S(++)) were derived from Eyring plots. In the absence of drugs, the activation enthalpy and the free energy of activation for P-glycoprotein ATPase activity was determined as Delta H(++) = 92.6 +/- 4.2 kJ/mol and Delta G(++) = 73.1 +/- 7.2 kJ/mol, respectively. Increasing the drug concentration reduced the activation enthalpy, whereby the drug with the highest transporter affinity had the strongest effect (DeltaDelta H(++) = -21%). The free energy of activation decreased for activating (DeltaDelta G(++) = approximately -3.8%) and increased for inhibitory compounds (DeltaDelta G(++) = approximately +0.7%). The drug-specific changes of the free energy of activation are thus barely above thermal energy. A comparison with literature data revealed that a decrease of the lateral membrane packing density reduces the enthalpic and the entropic contribution to the free energy of activation. Although the P-glycoprotein ATPase activity increases only slightly with decreasing lateral membrane packing density, the mode of action changes from strongly entropy-driven at high, to essentially enthalpy-driven at low packing densities. This suggests that the transporter and the membrane form a functional entity.     

P-glycoprotein effect on the properties of its natural lipid environment probed by Raman spectroscopy and Langmuir-Blodgett technique

Behavior of P-glycoprotein (Pgp) natural lipid environment within the membrane of CEM cells expressing Pgp in the quantities varying from 0% to 32% of the total amount of all membrane proteins is described for the first time. Observed cooperative effect of Pgp-induced increase of membrane stability, decrease of the temperature of gel-to-crystal lipids transition and predominance of the lipid liquid crystalline phase at physiological temperatures should have an impact in development of multidrug resistance phenotype of tumor cells by favoring the Pgp intercellular transfer and Pgp ATPase activity.     

On-line identification of P-glycoprotein substrates by monitoring of extracellular acidification and respiration rates in living cells

The influence of P-glycoprotein (ABCB1) in drug resistance as well as drug absorption and disposition is an important factor to be considered during the development of new drugs. Thus, the early identification and exclusion of compounds showing a high affinity towards P-glycoprotein can help to select drug candidates. The aim of our study was to implement a label-free assay for the identification of P-glycoprotein substrates in living cells. For this approach, a multiparametric, chip-based sensor system was used to determine extracellular acidification, cell respiration and adhesion upon stimulation with P-glycoprotein substrates. Using L-MDR1 cells, a human P-glycoprotein overexpressing cell line, the influence of P-glycoprotein activity was determined for seven different compounds, demonstrating the applicability of the system for P-glycoprotein substrate identification. Effects were concentration dependent, as shown for the P-glycoprotein substrate verapamil, and were associated with cellular acidification and respiration. P-glycoprotein ATPase activation by verapamil could be described by a Michaelis-Menten type kinetic profile showing saturation at high substrate concentrations. The Michaelis-Menten constants K(M) were determined to be 0.92μM (calculated based on extracellular acidification) and 4.9μM (calculated based on cellular respiration). Control experiments using 100nM of the P-glycoprotein inhibitor elacridar indicated that the observed effects were related to P-glycoprotein ATPase activity. In contrast, wild-type LLC-PK1 cells not expressing P-glycoprotein were not responsive towards stimulation with different P-glycoprotein substrates. Summarizing these findings, the used microsensor system is a generic system suitable for the identification of P-glycoprotein substrates. In contrast to biochemical P-glycoprotein assays, activation of the drug efflux pump can be monitored on-line in living cells to identify P-glycoprotein substrates and to study the molecular mechanisms of adenosintriphosphate-dependent active transport.     

Interaction of transported drugs with the lipid bilayer and P-glycoprotein through a solvation exchange mechanism

Broad substrate specificity of human P-glycoprotein (ABCB1) is an essential feature of multidrug resistance. Transport substrates of P-glycoprotein are mostly hydrophobic and many of them have net positive charge. These compounds partition into the membrane. Utilizing the energy of ATP hydrolysis, P-glycoprotein is thought to take up substrates from the cytoplasmic leaflet of the plasma membrane and to transport them to the outside of the cell. We examined this model by molecular dynamics simulation of the lipid bilayer, in the presence of transport substrates together with an atomic resolution structural model of P-glycoprotein. Taken together with previous electron paramagnetic resonance studies, the results suggest that most transported drugs are concentrated near the surface zone of the inner leaflet of the plasma membrane. Here the drugs can easily diffuse laterally into the drug-binding site of P-glycoprotein through an open cleft. It was concluded that the initial high-affinity drug-binding site was located in the interfacial surface area of P-glycoprotein in contact with the membrane interface. Based on these results and our recent kinetic studies, a “solvation exchange” drug transport mechanism of P-glycoprotein is discussed. A molecular basis for the improved colchicine transport efficiency by the much-studied colchicine-resistance G185V mutant human P-glycoprotein is also provided.     

Transport of (Glyco)Sphingolipids in and Between Cellular Membranes; Multidrug Transporters and Lateral Domains

Sphingolipids are highly enriched in the outer leaflet of the plasma membrane lipid bilayer. However, the first glycolipid, glucosylceramide, is synthesized in the opposite, cytosolic leaflet of the Golgi membrane. This has led us to experiments which suggest that the level of glucosylceramide in the cytosolic surface is carefully regulated both by the balance between synthesis and hydrolysis and by transport away from this surface through translocators, multidrug transporters, the same molecules that make cancer cells resistant to chemotherapy. Our data suggest a role for newly synthesized glucosylceramide not only in the formation of domains in the luminal leaflet of the Golgi but also on the cytosolic surface of this organelle.     

Transport of somatostatin and substance P by human P-glycoprotein

P-glycoprotein is an efflux pump for a broad spectrum of hydrophobic agents. We found that bioactive peptides including somatostatin and substance P inhibit ATP-dependent vincristine binding to P-glycoprotein-overexpressing K562/ADM membrane vesicles. Some of these bioactive peptides including somatostatin stimulate basal ATPase activity of P-glycoprotein; in contrast, other peptides including substance P inhibit it. The K562/ADM membrane vesicles showed an ATP-dependent, osmotically sensitive uptake of somatostatin and substance P, which was inhibited by valspodar, an inhibitor of P-glycoprotein. These findings suggested that certain bioactive peptides such as somatostatin and substance P directly interact with human P-glycoprotein as endogenous substrates for P-glycoprotein-mediated transport.     

Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter

The human ABCG2 multidrug transporter provides protection against numerous toxic compounds and causes multidrug resistance in cancer. Here we examined the effects of changes in membrane cholesterol on the function of this protein. Human ABCG2 was expressed in mammalian and in Sf9 insect cells, and membrane cholesterol depletion or enrichment was achieved by preincubation with beta cyclodextrin or its cholesterol-loaded form. We found that mild cholesterol depletion of intact mammalian cells inhibited ABCG2-dependent dye and drug extrusion in a reversible fashion, while the membrane localization of the transporter protein was unchanged. Cholesterol enrichment of cholesterol-poor Sf9 cell membrane vesicles greatly increased ABCG2-driven substrate uptake, substrate-stimulated ATPase activity, as well as the formation of a catalytic cycle intermediate (nucleotide trapping). Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. The selective, major effect of membrane cholesterol on the wild-type ABCG2 suggests a regulation of the activity of this multidrug transporter during processing or in membrane micro-domain interactions. The experimental recognition of physiological and pharmacological substrates of ABCG2, as well as the fight against cancer multidrug resistance may be facilitated by demonstrating the key role of membrane cholesterol in this transport activity.     

Phenothiazine maleates stimulate MRP1 transport activity in human erythrocytes

The expression of multidrug resistance-associated protein (MRP1) results in ATP-dependent reduction of drugs' concentration in cancer cells, i.e., multidrug resistance (MDR). Since the majority of projects are concentrated on the search of the new MDR modulators, there are very few reports on drug-induced stimulation of MDR transporters activity. In the present work, by means of functional fluorescence assay we have shown that MRP1-mediated efflux of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) out of human erythrocytes is stimulated by phenothiazine maleates that have been already identified as P-glycoprotein inhibitors. Phenothiazine maleates-induced stimulation of ATP-dependent uptake of 2',7'-bis-(3-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) into inside-out membrane vesicles prepared from erythrocyte membranes has been also demonstrated. Moreover, it was shown that phenothiazine maleates exerted stimulating effect on ATPase activity measured in erythrocyte membranes. To our best knowledge, this report is the first one demonstrating that compounds able to inhibit transport activity of P-glycoprotein can stimulate MRP1 transporter. We conclude that phenothiazine maleates probably exert their stimulatory effect on MRP1 by direct interaction with the protein at the site different from the substrate binding site.     

Effect of antimicrobial peptides on ATPase activity and proton pumping in plasma membrane vesicles obtained from mycobacteria

The potential usefulness of antimicrobial peptides (AMPs) as antimycobacterial compounds has not been extensively explored. Although a myriad of studies on AMPs from different sources have been done, some of its mechanisms of action are still unknown. Maganins are of particular interest since they do not lyse non-dividing mammalian cells. In this work, AMPs with well-recognized activity against bacteria were synthesized, characterized, purified and their antimycobacterial activity and influence on ATPase activity in mycobacterial plasma membrane vesicles were assessed. Using bioinformatics tools, a magainin-I analog peptide (MIAP) with improved antimicrobial activity was designed. The influence of MIAP on proton (H(+)) pumping mediated by F(1)F(0)-ATPase in plasma membrane vesicles obtained from Mycobacterium tuberculosis was evaluated. We observed that the antimycobacterial activity of AMPs was low and variable. However, the activity of the designed peptide MIAP against M. tuberculosis was 2-fold higher in comparison to magainin-I. The basal ATPase activity of mycobacterial plasma membrane vesicles decreased approximately 24-30% in the presence of AMPs. On the other hand, the MIAP peptide completely abolished the F(1)F(0)-ATPase activity involved in H(+) pumping across M. tuberculosis plasma membranes vesicles at levels similar to the specific inhibitor N,N' dicyclohexylcarbodiimide. These finding suggest that AMPs can inhibit the H(+) pumping F(1)F(0)-ATPase of mycobacterial plasma membrane that potentially interferes the internal pH and viability of mycobacteria.     

Upregulation of the expression of endogenous Mdr1 P-glycoprotein enhances lipid translocation in MDCK cells transfected with human MRP2

Various ABC transporters can translocate lipid molecules from the cytoplasmic into the exoplasmic leaflet of the plasma membrane bilayer. Two of these, MDR1 P-glycoprotein (Pgp) and MRP1, are multidrug transporters responsible for the resistance of various cancers against chemotherapy. We wanted to study whether MRP2, an ABC transporter of the bile canalicular membrane with a substrate specificity very similar to that of MRP1, is capable of translocating lipids. The translocation of short-chain lipids across the apical membrane of MDCK cells transfected with MRP2 was significantly higher than that in untransfected controls. However, the characteristics of the lipid translocation were similar to substrate transport by MDR1 and not MRP2: transport was strongly inhibited by classic MDR1 Pgp inhibitors, was independent of cellular glutathione, and was insensitive to a drug known to inhibit MRP2 activity. When tested by immunoblot, the MRP2-transfected cells expressed high levels of MRP2 but also of endogenous Mdr1. The expression of Mdr1 was unstable during maintenance of the cell line and correlated with the rate of lipid translocation across the apical membrane. We conclude that the observed increase in lipid transport in the MDCK cells transfected with MRP2 is the consequence of the upregulation of the expression of endogenous Mdr1 and that careful characterization of endogenous Mdr1 expression is needed in studies aimed to identify substrates of plasma membrane transporters.     

The membrane lipid environment modulates drug interactions with the P-glycoprotein multidrug transporter.

The P-glycoprotein multidrug transporter functions as an ATP-driven efflux pump for a large number of structurally unrelated hydrophobic compounds. Substrates are believed to gain access to the transporter after partitioning into the membrane, rather than from the extracellular aqueous phase. The binding of drug substrates to P-glycoprotein may thus be modulated by the properties of the lipid bilayer. The interactions with P-glycoprotein of two drugs (vinblastine and daunorubicin) and a chemosensitizer (verapamil) were characterized by quenching of purified fluorescently labeled protein in the presence of various phospholipids. Biphasic quench curves were observed for vinblastine and verapamil, suggesting that more than one molecule of these compounds may bind to the transporter simultaneously. All three drugs bound to P-glycoprotein with substantially higher affinity in egg phosphatidylcholine (PC), compared to brain phosphatidylserine (PS) and egg phosphatidylethanolamine (PE). The nature of the lipid acyl chains also modulated binding, with affinity decreasing in the order egg PC > dimyristoyl-PC (DMPC) > dipalmitoyl-PC (DPPC). Following reconstitution of the transporter into DMPC, all three compounds bound to P-glycoprotein with 2-4-fold higher affinity in gel phase lipid relative to liquid-crystalline phase lipid. The P-glycoprotein ATPase stimulation/inhibition profiles for the drugs were also altered in different lipids, in a manner consistent with the observed changes in binding affinity. The ability of the drugs to partition into bilayers of phosphatidylcholines was determined. All of the drugs partitioned much better into egg PC relative to DMPC and DPPC. The binding affinity increased (i.e., the value of Kd decreased) as the drug-lipid partition coefficient increased, supporting the proposal that the effective concentration of the drug substrate in the membrane is important for interaction with the transporter. These results provide support for the vacuum cleaner model of P-glycoprotein action.     

Characterization and functional reconstitution of the multidrug transporter

P-Glycoprotein, the multidrug transporter, is isolated from the plasma membrane of CH^RC5 cells using a selective two-step detergent extraction procedure. The partially purified protein displays a high level of ATPase activity, which has a highK _M for ATP, is stimulated by drugs, and can be distinguished from that of other membrane ATPases by its unique inhibition profile. Delipidation completely inactivates ATPase activity, which is restored by the addition of fluid lipid mixtures. P-Glycoprotein was reconstituted into lipid bilayers with retention of both drug transport and ATPase activity. Proteoliposomes containing P-glycoprotein display osmotically sensitive ATP-dependent accumulation of³H-colchicine in the vesicle lumen. Drug transport is active, generating a stable 5.6-fold concentration gradient, and can be blocked by compounds in the multidrug resistance spectrum. Reconstituted P-glycoprotein also exhibits a high level of ATPase activity which is further stimulated by various drugs. P-Glycoprotein therefore functions as an active drug transporter with constitutive ATPase activity.     

Energy-dependent efflux of methotrexate in L1210 leukemia cells. Evidence for the role of an ATPase obtained with inside-out plasma membrane vesicles.

Earlier studies from our laboratory (Dembo, M., Sirotnak F. M., and Moccio, D. M. (1984) J. Membr. Biol. 78, 9-17) suggested that methotrexate (MTX) efflux from L1210 cells was mediated predominantly by an ATP-dependent, outwardly directed, mechanism. To examine this process further, we utilized predominantly (74%) inside-out plasma membrane vesicle preparations derived from an L1210 cell variant (L1210/R24) with 15-fold reduced Vmax for [3H]MTX influx. Efflux of [3H]MTX, under nonionic buffer conditions, in these inside-out membrane vesicles was temperature and ATP dependent (apparent Km = 0.40 +/- 0.06 mM), osmotically sensitive, and unaffected by protonophores. The presence of K+, Na+, Cl-, and HCO3- at their physiological concentrations had no effect on [3H]MTX efflux. Other triphosphonucleotides (GTP and CTP), but not a nonhydrolyzable analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), could also stimulate efflux, but to a lesser extent. Also, ATP gamma S and orthovanadate were potent inhibitors of ATP-dependent efflux of [3H]MTX. Other experiments revealed a system with low saturability for [3H]MTX during efflux (apparent Km = 46 +/- 7 microM), but extremely high capacity (106 +/- 15 pmol/min/mg protein), and a pH optimum in the range of 5.5-6. However, appreciable efflux was measured in the physiological range of pH 6.7-6.9. A number of inhibitors or copermeants for ATP-dependent [3H]MTX efflux in intact L1210 cells were inhibitors of ATP-dependent efflux in inside-out plasma membrane vesicles, including, cholate, bromosulfophthalein, verapamil, quinidine, and reserpine. These findings and other results showing that bromosulfophthalein will completely inhibit efflux are consistent with a role for an ATPase in [3H]MTX efflux, and suggest that the process under study is the bromosulfophthalein-sensitive, ATP-dependent route responsible for the majority of [3H]MTX efflux in intact L1210 cells.     

Sidedness of yeast plasma membrane vesicles and mechanisms of activation of the ATPase by detergents

The binding of concanavalin A and of fluorescein 5'-isothiocyanate indicate similar amount of right-side-out and inside-out vesicles in plasma membrane vesicles from either glucose-starved or glucose-fermenting yeast cells. These vesicles contain low-activity and high-activity states of the ATPase, respectively. Unmasking of latent active sites can explain the limited ATPase activation (about 2-fold) produced by several detergents on both kinds of vesicles. On the other hand, lysophosphatidic acid (oleoyl) produces a 7-fold activation of the ATPase in vesicles from glucose-starved cells. This effect is accompanied by a change in Km of the enzyme and probably reflects a direct action of the detergent on the ATPase. A similar activation and Km change can be obtained by sonication of the vesicles, although in this case soybean phospholipids are required for maximal activity. Apparently the low-activity state of the yeast plasma membrane ATPase can be activated not only by glucose metabolism 'in vivo' (mechanism unknown) but also by some detergents and physical treatments 'in vitro'. Experiments with purified ATPase from glucose-starved cells also indicate that lysophosphatidic acid (oleoyl) specifically activates the enzyme. These results suggest a note of caution on considering the usual interpretation of the effects of detergents on membrane enzymes, which only take into account the unmasking of latent active sites.     

Undirectional calcium and nucleotide fluxes in cardiac sarcoplasmic reticulum. II. Experimental results.

Unidirectional calcium influx and efflux were evaluated in cardiac sarcoplasmic reticulum (SR) by 45Ca-40Ca exchange at steady state calcium uptake in the absence of calcium precipitating anions. Calcium efflux was partitioned into a pump-mediated efflux and a parallel passive efflux by separately measuring passive efflux referable to the steady state. Unidirectional and net ATP-ADP fluxes were measured using [3H]-ATP----ADP and [3H]-ADP----ATP exchanges. Methods are presented that take into account changing specific activities and sizes of the nucleotide pools during the measurement of nucleotide fluxes. The contribution of competent and incompetent vesicles to the unidirectional and net nucleotide fluxes was evaluated from the specific activity of these fluxes in incompetent vesicles and from the fraction of vesicles that were incompetent. The results indicate that, in cardiac SR, unidirectional calcium fluxes are larger than the unidirectional nucleotide fluxes contributed by competent vesicles. Because the net ATPase rate of competent vesicles is similar to the parallel passive efflux, it appears that cardiac SR Ca-ATPase tightly couples ATP hydrolysis to calcium transport even at static head, with a coupling ratio near 1.0.     

Perturbation of the lipid phase of a membrane is not involved in the modulation of MRP1 transport activity by flavonoids

The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.     

Passive Ca2+ permeability of vesicular sarcolemmal preparations from myocardium

Vesicular preparations of sarcolemma isolated from rat myocardium possessed high ATPase (4.32 +/0 0.57 micromole/min per mg), adenylate cyclase (121 +/- 11 pmole/min per mg) and creatine kinase (1.74 +/- 0.35 micromole/min per mg) activities and a Na-Ca exchange activity specific for sodium. The ATPase activity was inhibited by digitoxigenin by 50-70% and was not changed by ouabain, EGTA, ionophore A23187 and oligomycin, thus showing the absence of mitochondrial and sarcoplasmic reticulum contaminations in the sarcolemmal preparations. The preparations consisted mostly of closed inside-out vesicles. The preparation was used to study the mechanism of Ca2+ penetration across the sarcolemmal membrane. For this purpose the vesicles were load with 45Ca2+, which relatively slowly diffused from the medium into the vesicles, and which was bound to the binding sites inside the vesicles (n = 20.5 +/- 4.6 nmoles per mg of protein, Kd approximately equal to 1.8 +/- 0.21 mM). The transmembrane movement of Ca2+ was demonstrated by the following findings: 1) the ionophore A23187 only insignificantly increased the total vesicular Ca2+ content, but strongly accelerated Ca2+ efflux from the vesicles along its concentration gradient; 2) gramicidin and osmotic shock caused a similar acceleration of Ca2+ efflux. Ca2+ efflux from these vesicles along Ca2+ concentration gradient was studied under conditions, when the extravesicular Ca2+ content was lowered due to its binding to EGTA and by dilution. The gradient of Ca2+ concentration was from 2.0 mM inside to approximately 0.1 micro M outside. The rate of 45Ca2+ efflux depended hyperbolically on the intravesicular Ca2+ efflux from the vesicles was inhibited by Mn2+, Co2+ and verapamil when they acted from the inside of the vesicles. An increase in ionophore A23187 concentration increased the efflux of Ca2+ hyperbolically and enhanced only the maximal rate of the efflux. It is concluded that the passive permeability of Ca2+ across the sarcolemmal membrane along its concentration gradient is controlled by Ca2+ binding to the membrane.