Mechanism of active shrinkage in mitochondria I. Coupling between weak electrolyte fluxes

1. A passive penetration of (NH₄)₂ HPO₄ or of K₂HPO₄+nigericin occurs in respiratory-inhibited liver mitochondria. Addition of succinate at the end of the passive swelling initiates a shrinkage phase which leads to restoration of the initial mitochondrial volume. The rate and time of onset of the active shrinkage depend on the degree of stretching of the mitochondrial membrane. The rate of active shrinkage increases proportionally to the concentration of nigericin while it is strongly inhibited by valinomycin.2. A number of SH inhibitors such as N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuriphenylsulphonate, dithiobisnitrobenzoate, exert a marked enhancing effect on the rate of shrinkage. The enhancing effect parallels titration of the phosphate carrier and inhibition of the passive phosphate influx. The above SH inhibitors do not inhibit passive phosphate efflux. In contrast, mersalyl is a powerful inhibitor of the rate of active shrinkage. The inhibition parallels that on phosphate passive efflux and requires higher mersalyl concentrations in respect to inhibition of phosphate influx.3. The active shrinkage is discussed in terms of (a) a mechanoenzyme, (b) an electrogenic proton pump and (c) a proton-driven P_i pump.     

Mechanism of active shrinkage in mitochondria. II. Coupling between strong electrolyte fluxes.

1. Addition of succinate to valinomycin-treated mitochondria incubated in KCL causes a large electrolyte penetration. The process depends on a steady supply of energy and involves a continuous net extrusion of protons. Rates of respiration and of electrolyte penetration proceed in a parallel manner. 2. A passive penetration of K+ salt of permeant anions occurs in respiratory-inhibited mitochondria after addition of valinomycin. Addition of succinate at the end of the passive swelling starts an active extrusion of anions and cations with restoration of the initial volume. The shrinkage is accompanied by a slow reuptake of protons. The initiation of the active shrinkage correlates with the degree of stretching of the inner membrane. The extrusion of electrolytes is inhibited by nigericin, while it is only slightly sensitive to variations of the valinomycin concentration larger than two orders of magnitude. 3. Passive swelling and active shrinkage occurs also when K+ is replaced by a large variety of organic cations. The rate of organic cation penetration is enhanced by tetraphenylboron, while the rate of electrolyte extrusion is insensitive to variation of the tetraphenylboron concentration. 4. Active shrinkage, either with K+ or organic cation salts, is inhibited by weak acids. The phosphate inhibition is removed by SH inhibitors. The active shrinkage is also inhibited by mersalyl to an extent of about 60%. 5. Three models of active shrinkage are discussed: (a) mechanoprotein, (b) electrogenic proton pump, and (c) proton-driven cation anion pump.     

Mechanism of active shrinkage in mitochondria II. Coupling between strong electrolyte fluxes

1. Addition of succinate to valinomycin-treated mitochondria incubated in KCl causes a large electrolyte penetration. The process depends on a steady supply of energy and involves a continuous net extrusion of protons. Rates of respiration and of electrolyte penetration proceed in a parallel manner.2. A passive penetration of K⁺ salt of permeant anions occurs in respiratory-inhibited mitochondria after addition of valinomycin. Addition of succinate at the end of the passive swelling starts an active extrusion of anions and cations with restoration of the initial volume. The shrinkage is accompanied by a slow reuptake of protons. The initiation of the active shrinkage correlates with the degree of stretching of the inner membrane. The extrusion of electrolytes is inhibited by nigericin, while it is only slightly sensitive to variations of the valinomycin concentration larger than two orders of magnitude.3. Passive swelling and active shrinkage occurs also when K⁺ is replaced by a large variety of organic cations. The rate of organic cation penetration is enhanced by tetraphenylboron, while the rate of electrolyte extrusion is insensitive to variation of the tetraphenylboron concentration.4. Active shrinkage, either with K⁺ or organic cation salts, is inhibited by weak acids. The phosphate inhibition is removed by SH inhibitors. The active shrinkage is also inhibited by mersalyl to an extent of about 60%.5. Three models of active shrinkage are discussed: (a) mechanoprotein, (b) electrogenic proton pump, and (c) proton-driven cation anion pump.     

Phosphate-induced efflux of adenine nucleotides from rat-heart mitochondria: evaluation of the roles of the phosphate/hydroxyl exchanger and the dicarboxylate carrier

Upon the addition of inorganic phosphate, isolated rat-heart mitochondria released endogenous adenine nucleotides. To elucidate the mechanism of this phosphate-induced efflux, we evaluated the relative roles of three inner mitochondrial membrane carriers: the adenine nucleotide translocase, the phosphate/hydroxyl exchanger, and the dicarboxylate carrier. Atractyloside (a specific inhibitor of the adenine nucleotide translocase) prevented this efflux, but did not inhibit mitochondrial swelling. Inhibitors of the phosphate/hydroxyl exchanger (200 microM n-ethylmaleimide and 10 microM mersalyl) did not inhibit phosphate-induced efflux. 200 microM mersalyl (which inhibited both the phosphate/hydroxyl exchanger and the dicarboxylate carrier) inhibited the rate of efflux approx. 65% Phenylsuccinate and 2-n-butylmalonate (inhibitors of the dicarboxylate carrier) partially inhibited phosphate-induced efflux and adenine nucleotide translocase activity. Mersalyl (200 microM) had no effect on adenine nucleotide translocase activity. Partial inhibition of the adenine nucleotide translocase by phenylsuccinate and butylmalonate could not explain the extent of inhibition of phosphate-efflux by these agents. Moreover, the rates of adenine nucleotide efflux in the presence of phenylsuccinate, butylmalonate, or mersalyl correlated well with the ability of these agents to inhibit succinate-supported respiration. We conclude that phosphate-induced efflux of adenine nucleotides from rat heart mitochondria occurs over the adenine nucleotide translocase, and that the site of action of the phosphate is not the phosphate/hydroxyl exchanger, but is likely the dicarboxylate carrier.     

Glutamate-supported calcium movements in rat liver mitochondria effects of anions and pH

Ca²⁺ efflux from rat liver mitochondria in the presence of glutamate is stimulated by a decrease in pH from 7.3 to 6.8 and the rate is dependent on the phosphate concentration. During Ca²⁺ (13 μm) uptake and release at low pH (+ phosphate), swelling is minimal, but a large oxidation of pyridine nucleotides and sustained membrane depolarization occurs. The depolarization (but not Ca²⁺ efflux) is reversed by ruthenium red. An absolute requirement for phosphate to support Ca²⁺ efflux is demonstrated by using acetate or lactate to support Ca²⁺ uptake (efflux is depressed at pH 6.8). Preincubation with mersalyl, to block phosphate movements, with subsequent phosphate addition preceeding Ca²⁺ uptake also inhibits efflux. β-Mercaptoethanol then stimulates efflux concomittent with membrane repolarization. Ca²⁺ efflux is not a simple result of collapse of ΔpH since nigericin inhibits phosphate transport and Ca²⁺ release. Following Ca²⁺ uptake at pH 6.8, respiratory inhibition occurs, but oxygen consumption coupled to ATP synthesis can be stimulated by succinate (+ rotenone). Addition of succinate allows reuptake of Ca²⁺, reduction of pyridine nucleotides, and repolarization of the membrane potential. Respiratory inhibition is also seen with nigericin, but no Ca²⁺ efflux is observed. Coupled respiration with glutamate is seen at pH 6.8 following Ca²⁺ uptake in the presence of lactate with subsequent addition of phosphate to promote Ca²⁺ efflux. We conclude that Ca²⁺ efflux is not a consequence of respiratory inhibition, but is mediated solely by phosphate movements. The inhibitory effect of Mg²⁺ on Ca²⁺ efflux is probably due to Mg²⁺-dependent inhibition of the Ca²⁺ diffusion potential so that the compensatory increase in ΔpH due to membrane depolarization does not occur and phosphate entry is slowed.     

Reactivity of the -SH groups of the mitochondrial phosphate carrier under native, solubilized and reconstituted conditions

The isolated and liposome-reconstituted mitochondrial phosphate carrier exhibits a sigmoidal inhibition curve by mersalyl, similar to that found with intact mitochondria. In contrast a hyperbolic inhibition curve is found (a) by titration of the soluble carrier with mersalyl before reconstitution in liposomes and (b) by titration of the reconstituted carrier with mersalyl after successively pretreatment of the mitochondria with low, non-inhibitory concentrations of mersalyl, excess N-ethylmaleimide and dithiothreitol. The inhibition of the reconstituted, but not of the soluble, phosphate carrier by mersalyl can be reversed by dithiothreitol. Cupric di(1,10-phenanthroline) inhibits the soluble but not the reconstituted phosphate carrier. The inhibited phosphate carrier can be reactivated by dithiothreitol in the soluble state but not after reconstitution in liposomes. The data support the previously suggested model of the phosphate carrier, assuming a dimer of two identical subunits for the active unit.     

The transport of sulphate and sulphite in rat liver mitochondria

1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO(2) or an exchange of the sulphite anion for hydroxyl ion(s).     

Action of spermine on phosphate transport in liver mitochondria

Spermine, at concentrations similar to those normally present in the cytosol of liver cells, facilitates the transport of phosphate into mitochondria and thus its accumulation within the matrix space. Both mersalyl and N-ethylmaleimide (NEM) inhibit phosphate influx either in the absence or in the presence of spermine. These inhibitors also inhibit, but only partially, the efflux from mitochondria of phosphate generated within the matrix space by the hydrolysis of ATP induced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or the valinomycin-K+ system. The inhibition of phosphate efflux by both mersalyl and NEM is almost completely removed, unlike that of phosphate influx, by spermine. The possibility that spermine may induce phosphate efflux by damaging mitochondrial membranes and consequently inducing an unspecific permeability to phosphate is excluded by the full restoration of transmembrane potential once FCCP has been removed by albumin. Since spermine does not react with either thiol groups or thiol group reagents, the simplest explanation of the reported results is that the pathway of phosphate efflux is distinct from that of phosphate influx.     

Ca(2+) efflux in mitochondria from the yeast Endomyces magnusii.

Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein. The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions. Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.     

Calcium efflux parallel to total phosphate retention in rat liver mitochondria

Phosphate efflux from uncoupled rat liver mitochondria was completely inhibited when mersalyl plus butylmalonate and ATP were added to a sucrose suspending medium. Despite the total retention of phosphate a calcium efflux was observed even in presence of ruthenium red. Under the above conditions no phosphate is transported in association with the ADP/ATP carrier. While mersalyl completely blocked the phosphate release induced by ruthenium red or EGTA from coupled mitochondria it only partially inhibited the CA2+-efflux. The inhibition of Ca2+ efflux was almost completely abolished in the presence of acetate. The existence of a co-transport of Ca2+ associated with phosphate is discussed.     

Parallel efflux of Ca2+ and Pi in energized rat liver mitochondria.

Addition of Ruthenium Red to energized rat liver mitochondria that have previously accumulated Ca2+ and phosphate from the external medium induces a parallel efflux of both these ions. Mersalyl or dithioerythritol, which decrease Ruthenium Red-insensitive Ca2+ efflux, also decrease phosphate efflux to the same extent. Conversely diazenedicarboxylic acid bis(NN-dimethylamide) (DDBA), which increases the Ruthenium Red-induced Ca2+ efflux concurrently increases phosphate release. Dithioerythritol and DDBA, reducing and oxidizing agents of thiol groups respectively, modify Ca2+ and Pi efflux without penetrating the mitochondrial inner membrane. Under all the adopted conditions the membrane potential is preserved. The release of resting respiration and the parallel efflux of Mg2+ and adenine nucleotides, events closely correlated to Ca2+ cycling, are equally prevented either by mersalyl, which inhibits phosphate transport, or dithioerythritol; DDBA has the opposite effect. These findings and the observation that suggest that Ca2+ and phosphate transport in energized liver mitochondria are closely related and dependent on the redox state of membrane-bound thiol groups.     

Proteins dealing with N-ethylmaleimide inhibition of pig heart mitochondrial phosphate transport system.

Our data clearly demonstrate that protective effect of phosphate and protective effect of mersalyl against NEM-inhibition of phosphate transport act at the level of two kinds of proteins. (1)Two major components are phosphate and nigericin NEM sensitive. According to our previous data [13] it has been also demonstrated that these two proteins components are valinomycin NEM sensitive (results not shown here) suggesting a relationship between these proteins and the energy linked proton translocation process. Relationships between these proteins and the phosphate translocation process are not evident and are under further investigations. (2) Two other insoluble major components localised at the level of the subparticular fraction are mersalyl NEM sensitive. We can suggest that these proteins are implicated in the translocation of phosphate in pig heart mitochondria.     

Phosphate transport and proteins with SH groups in rat liver mitochondria

Phosphate transport in rat liver mitochondria was studied by following [32P] phosphate uptake within physiological concentrations. Transport inhibition due to mersalyl and protection by mersalyl against N-ethylmaleimide measured in those conditions corresponded to earlier results obtained by the swelling technique. When mitochondria were incubated with [3H] N-ethylmaleimide in the presence of mersalyl, the radioactive labeling in proteins of particles obtained after sonication was decreased in all fractions, but three proteins were both highly alkylated and also highly protected by mersalyl (M.W. 48,000 - 36,000 - 31,000). Two of these (M.W. 36,000 and 31,000) were partially purified by ultrogel chromatography in the presence of sodium dodecyl sulfate. Furthermore, it was shown that both phosphate and nigericin diminished labeling by N-ethylmaleimide in the final supernatant fraction. Two proteins (M.W. 98,000 and 31,000) were significantly alkylated by [3H] N-ethylmaleimide and protected by phosphate and nigericin.     

Bidirectional fluxes of spermine across the mitochondrial membrane

The polyamine spermine is transported into the mitochondrial matrix by an electrophoretic mechanism having as driving force the negative electrical membrane potential (ΔΨ). The presence of phosphate increases spermine uptake by reducing ΔpH and enhancing ΔΨ. The transport system is a specific uniporter constituted by a protein channel exhibiting two asymmetric energy barriers with the spermine binding site located in the energy well between the two barriers. Although spermine transport is electrophoretic in origin, its accumulation does not follow the Nernst equation for the presence of an efflux pathway. Spermine efflux may be induced by different agents, such as FCCP, antimycin A and mersalyl, able to completely or partially reduce the ΔΨ value and, consequently, suppress or weaken the force necessary to maintain spermine in the matrix. However this efflux may also take place in normal conditions when the electrophoretic accumulation of the polycationic polyamine induces a sufficient drop in ΔΨ able to trigger the efflux pathway. The release of the polyamine is most probably electroneutral in origin and can take place in exchange with protons or in symport with phosphate anion. The activity of both the uptake and efflux pathways induces a continuous cycling of spermine across the mitochondrial membrane, the rate of which may be prominent in imposing the concentrations of spermine in the inner and outer compartment. Thus, this event has a significant role on mitochondrial permeability transition modulation and consequently on the triggering of intrinsic apoptosis.     

Effect of mersalyl on mitochondrial Mg++ flux

The mercurial mersalyl has little effect either on rapid Mg⁺⁺ binding by isolated rat liver mitochondria or on the total Mg⁺⁺ content of these organelles measured after 0.75 min of incubation at 20°C. The data do not support the previous suggestion that the increased permeability to K⁺ of mitochondria treated with mersalyl results from release of endogenous Mg⁺⁺. An increased pH-dependence of unidirectional Mg⁺⁺ flux into respiring rat liver mitochondria is suggested to arise indirectly from inhibition by mersalyl of pH shifts associated with exchanges of endogenous phosphate. In addition, mersalyl appears to have a stimulatory effect on Mg⁺⁺ influx. Mersalyl also increases the average rate of unidirectional efflux of endogenous Mg⁺⁺. The stimulatory effects of mersalyl on Mg⁺⁺ flux are similar to, although quantitatively less than, the previously reported effects of mersalyl on mitochondrial K⁺ flux.     

Partial purification and characterization of the phosphate transporter from bovine heart mitochondria.

A highly active phosphate transporter was extracted with octylglucoside from bovine heart submitochondrial particles that were first partially depleted of other membrane components. It was then partially purified by ammonium sulfate fractionation. After reconstitution of the transporter into liposomes prepared with a crude mixture of soybean phospholipids, the Pi/OH exchange, but not the Pi/Pi exchange, was stimulated three- to fourfold by valinomycin and nigericin in the presence of K+. Both Pi/OH and Pi/Pi exchange activities were sensitive to mercurials and other SH reagents. The rutamycin-sensitive ATPase complex from mitochondria was reconstituted together with the phosphate transporter and adenine nucleotide transporter into liposomes. After inhibition of externally located ATPase, the hydrolysis of ATP was sensitive to atractyloside and mersalyl.     

Interaction of mitochondrial phosphate carrier with fatty acids and hydrophobic phosphate analogs

Mitochondrial transporters, in particular uncoupling proteins and the ADP/ATP carrier, are known to mediate uniport of anionic fatty acids (FAs), allowing FA cycling which is completed by the passive movement of FAs across the membrane in their protonated form. This study investigated the ability of the mitochondrial phosphate carrier to catalyze such a mechanism and, furthermore, how this putative activity is related to the previously observed HgCl(2)-induced uniport mode. The yeast mitochondrial phosphate carrier was expressed in Escherichia coli and then reconstituted into lipid vesicles. The FA-induced H(+) uniport or Cl(-) uniport were monitored fluorometrically after HgCl(2) addition. These transport activities were further characterized by testing various inhibitors of the two different transport modes. The phosphate carrier was found to mediate FA cycling, which led to H(+) efflux in proteoliposomes. This activity was insensitive to ATP, mersalyl or N-ethylmaleimide and was inhibited by methylenediphosphonate and iminodi(methylenephosphonate), which are new inhibitors of mitochondrial phosphate transport. Also, the HgCl(2) induced Cl(-) uniport mediated by the reconstituted yeast PIC, was found to be inhibited by these reagents. Both methylenediphosphonate and iminodi(methylenephosphonate) blocked unidirectional Cl(-) uptake, whereas Cl(-) efflux was inhibited by iminodi(methylenephosphonate) and phosphonoformic acid only. These results suggest that a hydrophobic domain, interacting with FAs, exists in the mitochondrial phosphate carrier, which is distinct from the phosphate transport pathway. This domain allows for FA anion uniport via the phosphate carrier and consequently, FA cycling that should lead to uncoupling in mitochondria. This might be considered as a side function of this carrier.     

A protective function of superoxide dismutase during respiratory chain activity.

(1) Aerobic incubation of heart muscle submitochondrial particles in phosphate buffer after treatment with NADH causes a progressive and substantial inhibition of the NADH oxidation system. Succinate oxidation remains almost unaffected by NADH treatment. (2) The loss of NADH oxidase activity is due to an inhibition of the respiratory chain-linked NADH dehydrogenase. This inhibition of the enzyme is very similar to that caused by combination of the organic mercurial mersalyl with NADH dehydrogenase. (3) The inhibition of NADH oxidation is largely prevented by compounds that are known to react with superoxide ions (02-.), including superoxide dismutase, cytochrome c, tiron and Mn2+. EDTA also has a protective effect, but a number of other metal chelating agents, and several proteins, including catalase, are without effect. (4) It is concluded that the inhibition of NADH oxidation of NADH oxidation by superoxide ions or by mersalyl is reversible and is therefore not due to the loss of oxidoreduction components from the respiratory chain or to an irreversible change in protein conformation. (6) The function of mitochondrial superxide dismutase is discussed in relation to the key role of NADH dehydrogenase in energy-conserving reactions and the formation of hydrogen peroxide during mitochondrial oxidations.     

Importance of the flux of phosphate across the inner membrane of kidney mitochondria for the activation of glutaminase and the transport of glutamine

The effect of mersalyl, an inhibitor of phosphate transport across the inner mitochondrial membrane, was investigated on the uncoupled respiration of pig kidney mitochondria in the presence of glutamine as substrate and on the activity of the phosphate-dependent glutaminase in the intact organelles. In addition, the submitochondrial location of the enzyme was reinvestigated.

1. (1) It was found that mersalyl completely inhibits uncoupled respiration of the mitochondria in the presence of glutamine as substrate, whereas respiration with glutamate was not affected. The same amount of mersalyl which inhibits coupled oxidation of glutamine also inhibits coupled oxidation of glutamate and some other substrates.

2. (2) Mersalyl strongly inhibited the activation of glutaminase in intact mitochondria only in the presence of inhibitors of electron transport or of an uncoupler. The addition of a detergent prevented or fully released the inhibition. The effect of mersalyl was observed even when the mitochondria were pre-incubated with phosphate or incubated in the phosphate-free medium. If mersalyl and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were added 3 min after pre-incubation with phosphate the same intramitochondrial concentration of the anion as in control experiments was found, whereas the activity of glutaminase was severely inhibited. These findings suggest that the activation of the enzyme by phosphate in intact nonenergized mitochondria occurs only if the activator moves across the inner mitochondrial membrane.

3. (3) Mersalyl (plus CCCP) markedly decreased [¹⁴C]glutamine- and [³²P]-phosphate-permeable mitochondrial spaces. A close correlation between the decrease of phosphate and glutamine permeable spaces and the inhibition of glutaminase activity was found.

4. (4) If the activation energy of the enzyme was determined with frozen mitochondrial preparations, a discontinuity or break in the Arrhenius plot was observed, whereas the presence of a detergent completely abolished the break. Digitonin or ultrasonic treatment of the mitochondria followed by separation of the membrane and the soluble fraction revealed that glutaminase is a membrane-bound enzyme.

On the basis of these findings it is concluded that there is an association between the transport of phosphate on one side and the transport of glutamine and glutaminase activity on the other. It is possible that the movement of phosphate across the membrane activates the enzyme which facilitates diffusion of glutamine down a concentration gradient. However, the existence of a specific glutamine-phosphate carrier is not ruled out.     

Kinetics of daunorubicin transport by P-glycoprotein of intact cancer cells.

Drug permeation across the plasma membrane of multidrug-resistant cells depends on the kinetics of the P-glycoprotein-mediated pump activity as well as on the passive permeation of the drug. We here demonstrate a method to characterize kinetically the pump in intact cells. To this purpose, we examined the membrane-transport properties of daunorubicin in various sensitive cancer cell lines and in their multidrug resistant (MDR) counterparts. First, we determined the passive permeability coefficient for daunorubicin. Then, using a flow-through system, the drug flux into the cell was measured after inhibition of the P-glycoprotein-mediated efflux pump. Combining the two results allowed us to calculate the intracellular free concentration of the drug. In the steady-state, the pump rate must equal the net rate of passive diffusion of the drug and, therefore, the same experiments gave us the pumping rate of daunorubicin. These experiments were then repeated at various extracellular drug concentrations. By plotting the pumping rate versus the intracellular drug concentration, we then characterized the P-glycoprotein kinetically. Four independent methods were used to measure the passive permeability coefficient for the cell line A2780. Similar values were obtained. Maximal pump rates (Vmax) showed a good correlation with the amount of P-glycoprotein in the cell lines used. We obtained saturation curves for the variation of the pump rates with the intracellular daunorubicin concentrations. These curves were typical for positive cooperativity, which provides evidence that at least two binding sites for daunorubicin are present on the active transport system of daunorubicin. The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic daunorubicin concentrations at half-maximal velocity for the cell lines used, were approximately 1.5 microM. Except for the cell lines with the highest amount of P-glycoprotein, the passive efflux rate of daunorubicin proved to be a substantial part of the total daunorubicin efflux rate for the cell lines used. In cell lines with relatively low levels of P-glycoprotein, passive daunorubicin efflux was even the main route of daunorubicin transport from the cells, determining the intracellular steady-state concentrations of daunorubicin.