Verapamil competes with doxorubicin for binding to anionic phospholipids resulting in increased internal concentrations and rates of passive transport of doxorubicin.

It is well documented that the Ca2+ channel antagonist verapamil can reverse multidrug resistance in cancer cells by decreasing P-glycoprotein mediated drug efflux. However, less information is available about effects of verapamil on drug-phospholipid interactions and on passive diffusion of drugs across the membrane, which both may play an important role in resensitizing cells to anti-cancer drugs. Therefore we studied the binding of verapamil to model membranes (large unilamellar vesicles) composed of various phospholipids and biological membranes. An increase of the amount of anionic phospholipids resulted in an enhanced binding of verapamil. Competition between verapamil and the anti-cancer drug and P-glycoprotein substrate doxorubicin for binding to anionic phospholipids was observed in model membranes composed of synthetic lipids, or composed of native Escherichia coli phospholipid mixtures, and in cytoplasmic membrane vesicles of this organism. Furthermore, verapamil specifically increased the rate of passive diffusion of doxorubicin across model membranes containing anionic phospholipids. It can be concluded that besides the decrease of P-glycoprotein mediated efflux at least two other effects may account for an increase of the internal (free and DNA-bound) doxorubicin concentration in the presence of verapamil; (i) a decrease of binding to anionic phospholipids in plasma-and intracellular membranes and (ii) an increase of the rate of passive import of doxorubicin across the plasma membrane.

Transport of the anti-cancer drug doxorubicin across cytoplasmic membranes and membranes composed of phospholipids derived from Escherichia coli occurs via a similar mechanism.

An assay was developed to measure and directly compare transport of doxorubicin across right-side-out cytoplasmic membrane vesicles (ROV) and across model membranes (LUVET) composed of pure phospholipids, isolated from the corresponding cells. Escherichia coli was used as a model organism, since mutants are available which differ in phospholipid composition. Both in LUVET and ROV only passive diffusion across the bilayer is involved, because effects of drug concentration, pH, divalent cations, the phospholipid composition, and the active transport inhibitor verapamil were comparable. Permeability coefficients were about 2-3-times higher in ROV compared to LUVET. Furthermore, in LUVET an average activation energy of 87 kJ/mol and in ROV of 50 kJ/mol was observed. These differences are suggested to result from differences in membrane order between LUVET and ROV and differences in the temperature dependence of membrane order in LUVET and ROV, respectively. Because no background carrier-facilitated doxorubicin transport seems to be present, ROV are an excellent model system to study the effect of phospholipid composition on drug transport after expression of a multidrug resistance-conferring protein. Furthermore, data of passive diffusion of doxorubicin obtained with LUVET are representative for more complex, biologically relevant membrane systems.

The interaction of the anti-cancer drug cisplatin with phospholipids is specific for negatively charged phospholipids and takes place at low chloride ion concentration.

The interaction of the anti-cancer drug cis-diamminedichloroplatinum(II) (cisPt) with model membranes was studied, with emphasis on the cisPt and phospholipid species involved. Binding studies using large unilamellar vesicles have revealed that: (i) Interaction involved negatively charged phospholipids only, and (ii) Interaction with negatively charged phospholipids was observed only in buffers with low Cl- concentration, indicating that aquated, positively charged cisPt is involved. Binding to all negatively charged phospholipids tested was highest at pH 6.0. At pH 7.4 a high and specific binding was observed with phosphatidic acid and phosphatidylserine. The consequences of cisPt binding on the organization of lipids was investigated with differential scanning calorimetry studies. These studies have indicated a higher ordering of dispersions of negatively charged phospholipids in the presence of divalent cationic cisPt. Summarizing, the interaction of positively charged cisPt species with negatively charged phospholipids is significant and should be considered in in vivo experiments.

The anionic phospholipid-mediated membrane interaction of the anti-cancer drug doxorubicin is enhanced by phosphatidylethanolamine compared to other zwitterionic phospholipids.

The interaction of doxorubicin and lipids has been studied using large unilamellar vesicles (LUVET) composed of mixtures of anionic phospholipids and various zwitterionic phospholipids. Dilution of anionic lipids with zwitterionic lipids leads to decreased membrane association of the drug because electrostatic forces are very important in doxorubicin-membrane interaction. However, binding of doxorubicin to LUVET composed of anionic phospholipids combined with phosphatidylethanolamine (PE) is much higher than binding to LUVET made of anionic lipids plus a range of other zwitterionic lipids such as phosphatidylcholine (PC) and the N-methylethanolamine and N, N-dimethylethanolamine derivatives of PE. This preferential interaction is observed with all negatively charged phospholipids tested and is, in the case of phosphatidylserine (PS), confirmed in monolayer experiments. The increase in surface area observed in a monolayer composed of PS and PE (1/3) was 3 times higher than in a monolayer of PS/PC (1/3). The preferential interaction appears not to be due to the ability of PE to adopt inverted nonbilayer structures, but probably involves a combination of the ability of PE to form additional hydrogen bonds and of the intrinsic curvature of a bilayer containing PE because of its small headgroup. Implications of our finding for the in vivo membrane interaction and transport of the drug will be discussed.

Energy transduction in the thermophilic anaerobic bacterium Clostridium fervidus is exclusively coupled to sodium ions.

The thermophilic, peptidolytic, anaerobic bacterium Clostridium fervidus is unable to generate a pH gradient in the range of 5.5-8.0, which limits growth of the organism to a narrow pH range (6.3-7.7). A significant membrane potential (delta psi approximately -60 mV) and chemical gradient of Na+ (-Z delta pNa approximately -60 mV) are formed in the presence of metabolizable substrates. Energy-dependent Na+ efflux is inhibited by the Na+/H+ ionophore monensin but is stimulated by uncouplers, suggesting that the Na+ gradient is formed by a primary pumping mechanism rather than by secondary Na+/H+ antiport. This primary sodium pump was found to be an ATPase that has been characterized in inside-out membrane vesicles and in proteoliposomes in which solubilized ATPase was reconstituted. The enzyme is stimulated by Na+, resistant to vanadate, and sensitive to nitrate, which is indicative of an F/V-type Na(+)-ATPase. In the proteoliposomes Na+ accumulation depends on the presence of ATP, is inhibited by the ATPase inhibitor nitrate, and is completely prevented by the ionophore monensin but is stimulated by protonophores and valinomycin. These and previous observations, which indicated that secondary amino acid transport uses solely Na+ as coupling ion, demonstrate that energy transduction at the membrane in C. fervidus is exclusively dependent on a Na+ cycle.

The F- or V-type Na(+)-ATPase of the thermophilic bacterium Clostridium fervidus.

Clostridium fervidus is a thermophilic, anaerobic bacterium which uses solely Na+ as a coupling ion for energy transduction. Important features of the primary Na+ pump (ATPase) that generates the sodium motive force are presented. The advantage of using a sodium rather than a proton motive force at high temperatures becomes apparent from the effect of temperature on H+ and Na+ permeation in liposomes.

Na(+) as coupling ion in energy transduction in extremophilic Bacteria and Archaea.

For microorganisms to live under extreme physical conditions requires important adaptations of the cells. In many organisms the use of Na(+) instead of protons as coupling ion in energy transduction is associated with such adaptation. This review focuses on the enzymes that are responsible for the generation and utilization of Na(+) gradients in extremophilic microorganisms. Aspects that are dealt with include: bioenergetics and ion homeostasis in extremophilic Bacteria and Archaea; the molecular mechanism of Na(+) translocation; and (dis)advantages of Na(+) as coupling ion in energy transduction.

Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus.

Amino acid transport was studied in membrane vesicles of the thermophilic anaerobic bacterium Clostridium fervidus. Neutral, acidic, and basic as well as aromatic amino acids were transported at 40 degrees C upon the imposition of an artificial membrane potential (delta psi) and a chemical gradient of sodium ions (delta microNa+). The presence of sodium ions was essential for the uptake of amino acids, and imposition of a chemical gradient of sodium ions alone was sufficient to drive amino acid uptake, indicating that amino acids are symported with sodium ions instead of with protons. Lithium ions, but no other cations tested, could replace sodium ions in serine transport. The transient character of artificial membrane potentials, especially at higher temperatures, severely limits their applicability for more detailed studies of a specific transport system. To obtain a constant proton motive force, the thermostable and thermoactive primary proton pump cytochrome c oxidase from Bacillus stearothermophilus was incorporated into membrane vesicles of C. fervidus. Serine transport could be driven by a membrane potential generated by the proton pump. Interconversion of the pH gradient into a sodium gradient by the ionophore monensin stimulated serine uptake. The serine carrier had a high affinity for serine (Kt = 10 microM) and a low affinity for sodium ions (apparent Kt = 2.5 mM). The mechanistic Na+-serine stoichiometry was determined to be 1:1 from the steady-state levels of the proton motive force, sodium gradient, and serine uptake. A 1:1 stoichiometry was also found for Na+-glutamate transport, and uptake of glutamate appeared to be an electroneutral process.

Amino acid transport in the thermophilic anaerobe Clostridium fervidus is driven by an electrochemical sodium gradient.

Amino acid transport was studied in membranes of the peptidolytic, thermophilic, anaerobic bacterium Clostridium fervidus. Uptake of the negatively charged amino acid L-glutamate, the neutral amino acid L-serine, and the positively charged amino acid L-arginine was examined in membrane vesicles fused with cytochrome c-containing liposomes. Artificial ion diffusion gradients were also applied to establish the specific driving forces for the individual amino acid transport systems. Each amino acid was driven by the delta psi and delta mu Na+/F and not by the Z delta pH. The Na+ stoichiometry was estimated from the amino acid-dependent 22Na+ efflux and Na(+)-dependent 3H-amino acid efflux. Serine and arginine were symported with 1 Na+ and glutamate with 2 Na+. C. fervidus membranes contain Na+/Na+ exchange activity, but Na+/H+ exchange activity could not be demonstrated.

Transport studies of doxorubicin in model membranes indicate a difference in passive diffusion across and binding at the outer and inner leaflets of the plasma membrane.

The kinetics of passive transport of the anticancer drug doxorubicin were analyzed in relation to membrane composition in large unilamellar vesicles in which DNA was enclosed. Special attention was paid to lipids that are typical for the inner and outer leaflet of the plasma membrane of mammalian cells: Phosphatidylethanolamine and anionic phosphatidylserine versus phosphatidylcholine, sphingomyelin, and cholesterol, respectively. The presence of anionic phospholipids results in a highly efficient incorporation of the drug into biological and model membranes [de Wolf, F. A., et al. (1993) Biochemistry 32, 6688-6695]. Therefore, the effect of drug binding on the amount of free, transportable drug was explicitly taken into account. However, even after correction for binding the permeability coefficient was about 35% lower in membranes containing 50 mol % of the anionic phosphatidylserine than in membranes consisting only of zwitterionic phospholipids (0.71-0.79 versus 1.18-1.25 microns s-1). This shows that drug binding and insertion also affect the intrinsic transport characteristics of the membranes. As compared to pure phosphatidylcholine, binding was not influenced by the incorporation of sphingomyelin or cholesterol, but equimolar amounts of sphingomyelin and cholesterol in phosphatidylcholine membranes decreased the rate of doxorubicin transport by 60% and 80%, respectively. The inhibitory effect of these two lipids is probably due to a closer packing of the membranes. In accordance, after the acyl chain order was decreased by adding the anaesthetic-like phenethyl alcohol (0.5% v/v), transport was stimulated more than 4-fold. The implications of our findings for the functioning and rate of drug pumping by the multidrug resistance-conferring P-glycoprotein in cancer cells are discussed.

Cisplatin complexes with phosphatidylserine in membranes.

Upon incubation of the anticancer drug cisplatin [cis-diamminedichloroplatinum(II)] with model membranes composed of phosphatidylserine (PS), a stable product is formed that has been isolated after chloroform/methanol extraction of the sample. The product formation is specific for PS and does not occur with other major membrane phospholipids. The rate and extent of product formation is dependent on the pH, chloride ion concentration, and temperature, with the highest rate at pH 6.0, in the absence of Cl- and at 37 degrees C, indicating that positively charged aquated cisplatin is the reactive species. Over 80% of PS is converted within 15 h under these conditions with a halftime of 5 h. PS can be regenerated by an excess of glutathione. Mass spectrometry experiments demonstrate that interaction of cisplatin with PS involves a loss of two chloride ions and coordination of platinum to the amine and carboxyl group of the serine moiety. Cisplatin forms complexes specifically with PS not only in model membranes but also in the plasma membrane of human erythrocytes. Since PS is essential in several cellular processes, its interaction with cisplatin may have important physiological implications.

Energy transduction and transport processes in thermophilic bacteria.

Bacterial growth at the extremes of temperature has remained a fascinating aspect in the study of membrane function and structure. The stability of the integral membrane proteins of thermophiles make them particularly amenable to study. Respiratory enzymes of thermophiles appear to be functionally similar to the mesophilic enzymes but differ in their thermostability and unusual high turnover rates. Energy coupling at extreme temperatures seems inefficient as suggested by the high maintenance coefficients and the high permeability of the cell membrane to protons. Nevertheless, membranes maintain their structure at these extremes through changes in fatty acid acyl chain composition. Archaebacteria synthesize novel membrane-spanning lipids with unique physical characteristics. Thermophiles have adapted to life at extreme temperatures by using sodium ions rather than protons as coupling ions in solute transport. Genetic and biochemical studies of these systems now reveal fundamental principles of such adaptations. The recent development of reconstitution techniques using membrane-spanning lipids allows a rigorous biochemical characterization of membrane proteins of extreme thermophiles in their natural environment.