©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protonophoric Activity of Ellipticine and Isomers across the Energy-transducing Membrane of Mitochondria (*)

(Received for publication, January 24, 1995; and in revised form, June 13, 1995)

Marc-Antoine Schwaller (1)(§) Beatrice Allard (1) Elie Lescot (2) François Moreau (3)

From the  (1)Institut de Topologie et Dynamique des Systèmes, CNRS URA 34, Université Paris 7, 75005 Paris, the (2)Laboratoire de Pharmacologie Moléculaire, CNRS URA 147, INSERM U 140, Institut Gustave Roussy, 94805 Villejuif, and the (3)Laboratoire de Physiologie Cellulaire et Moléculaire, CNRS URA 1180, Université Pierre et Marie Curie, 75005 Paris, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ellipticine is an antitumor alkaloid capable of uncoupling mitochondrial oxidative phosphorylation. It behaves as a lipophilic weak base with pK = 7.40. We have investigated its molecular mode of action using several of its isomers with pK ranging between 5.8 and 7.7 and ellipticinium, which is a permanent cationic derivative. The effects of these molecules on mitochondrial oxygen uptake and transmembrane potential were compared at different pHs. Ellipticinium exhibited very low effects on both respiratory rate and membrane potential. By contrast, protonable derivatives showed maximal stimulation of oxygen uptake and depolarizing effects when the pH of the medium was close to the drug pK. These effects were lowered when the transmembrane DeltapH was dissipated, which indicates that the neutral form of the drug is implicated in the uncoupling mechanism. In addition, protonable derivatives of ellipticine display a linear relationship between oxidation rate and transmembrane potential, which suggests that the uncoupling properties of these molecules result from a protonophoric mechanism. From these results, the following cyclic protonophoric mechanism is proposed for protonable ellipticines: (i) electrophoretical accumulation of the protonated form; (ii) deprotonation at the matrix interface; (iii) diffusion outwards; and (iv) reprotonation at the external interface.


INTRODUCTION

Ellipticine, a natural alkaloid from Ochrosia elliptica, has been shown to display some antitumor properties (1) . A large number of ellipticine derivatives have been synthesized (2) to investigate its mode of action. It has been shown that this molecule is able to intercalate between DNA base pairs(3) , and some authors have shown a good in vitro correlation between cytotoxicity and induced dichroic spectra of drug-DNA complexes(4) . However, good evidence lacks when it comes to correlating in vivo activities with a specific DNA binding mode(5) . Data based on microspectrofluorometric analyses have shown that an important fraction of ellipticine accumulates in mitochondria(6) . The possibility that these organelles rather than the nucleus might be a privileged pharmacological target has been suggested. This assumption has been supported by data indicating that ellipticine is a potent inhibitor of electron transfer in mitochondrial membranes. It has been shown that low concentrations of ellipticine uncouple oxidative phosphorylation. At higher concentrations, the drug inhibits the transfer of electrons at the level of cytochrome c oxidase (7) , which is likely to severely affect the cell viability through ATP deprivation. It can be mentioned that similar mitochondrial interactions were also observed with other antitumor DNA intercalating drugs, particularly in the anthracyclin series(8, 9) .

In a previous paper, we had investigated the protonation state of ellipticine bound to mitochondrial membranes at low drug concentrations that do not affect mitochondrial functions(10) . In these conditions, ellipticine was mainly detected within the inner mitochondrial membrane, and its protonation equilibrium was shown to directly reflect the electrogenic H ion movement that occurs during energy coupling(10) . In the present work we have investigated the effect of concentrations of ellipticine that are high enough to uncouple mitochondrial oxidative phosphorylation. The effect of ellipticine on oxygen uptake was investigated at different pHs in the absence or in the presence of nigericin to determine the influence of the transmembrane DeltapH. In addition we have investigated the effect of the pK of the drugs on both mitochondrial oxygen uptake and transmembrane electrical potential at a constant pH. We have also compared the effects of ellipticine with its N-methyl derivative ellipticinium, which behaves as a permanent cation(11) . Based on the data obtained, a new cyclic protonophoric mechanism is proposed for ellipticine and its isomers. This mechanism is discussed according to the physicochemical properties of these molecules.


MATERIALS AND METHODS

Chemicals

The chemical structure of ellipticine isomers and derivative are presented in Fig. 1. Ellipticine 1 and isoellipticine 3 were synthesized according to previously described procedures(13, 14) . The synthesis of isomers 5 and 6 has been recently described(15, 16) . Olivacin 4 was a generous gift from Prof. H. P. Husson (Université René Descartes, Paris). Ellipticinium 2 was obtained by quaternarization of ellipticine with methyl iodide in N,N-dimethylformamide. The drugs were routinely checked for purity by both thin layer chromatography and high pressure liquid chromatography. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), (^1)nigericin, ADP, valinomycin, antimycin A, sodium ascorbate, N,N,N`,N`-tetramethyl-p-phenylenediamine, and bovine serum albumin were purchased from Sigma. Safranine was obtained from Janssen and used without further purification. Tetraphenylphosphonium (TPP) was purchased from Merck.


Figure 1: Chemical formula of ellipticine 1, isomers 3-6, and the derivative ellipticinium 2. The drug pK of ellipticine 1 and isomers 3, 4, 5, and 6 are 7.4, 6.7, 7.7, 7.1, and 5.8, respectively.



Mitochondria Isolation

Rat liver mitochondria from male Wistar albino rats were isolated by differential centrifugation as described previously(6) .

Oxygen Uptake Measurements

Oxygen uptake was determined by polarography at 25 °C using a DW-1 Clark-type electrode from Hansatech (Hansatech Ltd., Kings Lynn, UK). The final volume was 1 ml of medium containing 0.25 M sucrose, 5 mM MgCl(2), 1 mM EDTA, 10 mM KCl, 10 mM KH(2)PO(4), and 10 mM HEPES buffer, pH 7.3. For the overall segment succinate-O(2), mitochondria (0.5 mg of protein/ml) were incubated using 15 mM succinate as a substrate. Ellipticine, isomers, or derivative were added after a first transition state 3/state 4 triggered by the addition of a limiting amount of ADP. For the segment cytochrome c-O(2), mitochondria (0.5 mg of protein/ml) were incubated in the presence of 2.5 mM ascorbate and 0.1 mMN,N,N`,N`-tetramethyl-p-phenylenediamine as substrate. Antimycin A (1 µg/mg protein) was added in order to inhibit electron transport in the span from cytochrome b to cytochrome c.

Membrane Potential Measurements

Variations in membrane potential were measured by using safranine as an optical probe(17) . Membrane potential and oxygen uptake were measured simultaneously using a TPP-sensitive electrode(18) . The concentration of TPP in the assay medium was 5 µM. After calibration of the electrode(19) , mitochondria (0.5-1 mg of protein) and succinate were added. Ellipticine, isomers, and derivative were added after a first transition state 3/state 4 triggered by the addition of a limiting amount of ADP.


RESULTS

Effect on Oxygen Uptake

The addition of ellipticine has two types of effects on the uptake of oxygen by mitochondria, depending on the concentration (Fig. 2A). At concentrations lower than 100 nmolbulletmg protein, state 4 respiration was stimulated, whereas it was inhibited for higher drug concentrations. This suggests that the main effect of the drug at low concentrations is to uncouple oxidative phosphorylation, although it mainly inhibits electron transfer activities at high concentrations. When the inhibitory effect of ellipticine was investigated using a suspension of uncoupled mitochondria (1 µM CCCP), it was detected for concentrations higher than 100 nmolbulletmg protein. As already shown(7) , the inhibitory effect occurs at the level of the cytochrome oxidase (ascorbate + N,N,N`,N`-tetramethyl-p-phenylenediamine oxidation, Fig. 2A).


Figure 2: Effects of ellipticine, ellipticinium, and isomers on the stimulation of oxygen uptake by rat liver mitochondria at pH 7.3. A, variation of oxygen uptake in mitochondria (0.5 mg of protein/ml)-oxidizing succinate (span succinate-O(2)) in the absence () or in the presence (up triangle) of 1 µM CCCP or ascorbate (+N,N,N`,N`-tetramethyl-p-phenylenediamine) (span cytochrome c-O(2)) upon the addition of increasing concentrations of ellipticine (box). B, effect of increasing concentrations of ellipticine (), ellipticinium (), or isomers 3 (+), 4 (circle), 5 (), or 6 (bullet) on oxygen uptake in mitochondria-oxidizing succinate. Experimental conditions are described under ``Materials and Methods.''



The effect of ellipticine isomers and derivative on O(2) uptake was also investigated (Fig. 2B). Except for the quaternarized ellipticinium derivative(2) , which displays very low stimulation, all isomers markedly stimulate O(2) uptake for concentrations lower than 300 nmolbulletmg protein. The stimulatory effect reaches a maximum value of 300% in the case of isomer 5. Protonable isomers do not display any simple correlation between the drug pK and the stimulatory effect. Actually, isomers with lowest pK values (3 and 6) and those with highest pK values (1 and 4) exhibited the lowest stimulatory effects. This result indicates that the effect of the drugs on O(2) uptake may not be specifically attributed to either the cationic or the neutral form but seems to result from the coexistence of the two forms.

The relationship between the drug protonation equilibrium and the effects on respiratory rate were further investigated by studying the influence of the incubation mixture pH on O(2) uptake (Fig. 3). Concentrations of the drugs used in these experiments are the ones that led to a maximal respiratory stimulation (see Fig. 2). For each protonable isomer, an optimal pH leading to a maximal stimulation was determined. These optimal pH values were found to be close to the drug pKs (Fig. 1).


Figure 3: Effect of pH on oxygen uptake in mitochondria-oxidizing succinate in the presence of optimal concentration of ellipticine 1 and isomers 3-5. Conditions are those described in the legend to Fig. 2. Concentration of ellipticine (), isomers 3 (+), 4 (circle), and 5 () are 244, 68, 230, and 154 µM, respectively.



Because a positive transmembrane DeltapH exists in respiring mitochondria, the protonation equilibrium of the ellipticine isomers may vary as a function of their localization. Stimulation of O(2) uptake by ellipticine was therefore investigated at three different pHs in the absence or in the presence of nigericin, a protonophore that collapses the transmembrane DeltapH. As shown in Fig. 4, when mitochondria are incubated with ellipticine (90 nmolbulletmg protein), the addition of nigericin clearly reduces the stimulation in O(2) uptake. This indicates that the presence of a mitochondrial transmembrane DeltapH increases the uncoupling activity of the drug. This observation together with the previously noted closeness of the optimal respiratory stimulation to the drug pKs strongly suggests that the occurrence of a deprotonation step in the matrix is involved in the uncoupling mechanism.


Figure 4: Effect of nigericin on the stimulation of oxygen uptake by ellipticine at three different pHs. Ellipticine (50 µM) was added to mitochondria (0.5 mg of protein/ml)-oxidizing succinate in the absence (hatched bars) or in the presence (stippled bars) of 0.1 µg of nigericin/mg of protein.



Effect on Membrane Potential

To precisely define the role of the pK of the molecules on their mode of action, we have investigated the effect of these drugs on mitochondrial membrane potential at constant pH (pH 7.3). For this purpose, membrane potential and O(2) uptake were measured either simultaneously using a TPP-sensitive electrode or independently using safranine as an optical probe. Both methods provide similar results for ellipticine and isomers 3-6. For ellipticinium, the membrane potential was measured with safranine only, because this drug interacts with the TPP-sensitive electrode. The addition of the drugs led to a depolarization of the inner mitochondrial membrane for concentrations that either stimulate or inhibit the respiration (Fig. 5). Ellipticine and isomer 5 display the highest effects on membrane potential, whereas the permanent cation ellipticinium was less efficient. In all cases (Fig. 6), the maximal depolarization is obtained with drugs whose pK values are close to the pH of the incubation mixture.


Figure 5: Effect of increasing concentrations of ellipticine 1, ellipticinium 2, and isomers 3-6 on mitochondrial transmembrane potential. Ellipticine (), ellipticinium (), and isomers 3 (+), 4 (circle), 5 (), and 6 (bullet) were added to mitochondria (0.5 mg of protein/ml)-oxidizing succinate at pH 7.3. Membrane potential was measured as described under ``Materials and Methods'' using the TPP-sensitive electrode or the safranine method.




Figure 6: Dependence of depolarization of mitochondrial membranes on the pK of ellipticine 1 and isomers 3-6. Data were taken from Fig. 5. pK values of ellipticine (), isomers 3 (+), 4 (circle), 5 (), and 6 (bullet) are 7.4, 6.7, 7.7, 7.1, and 5.8, respectively. Drug concentrations are 38 (lower curve) and 154 µM (upper curve).



The relationship between the oxidation rate and the membrane potential was further analyzed for ellipticine and its isomers, using CCCP as a reference protonophore (Fig. 7). It is well established that for classical protonophoric uncouplers like CCCP, a linear relationship between the oxidation rate and membrane potential is obtained(19, 20) . The same linear relationship was observed for ellipticine and its isomers in the range of concentrations that led to a stimulatory effect on electron transfer (Fig. 7). In the case of isomer 3, which displays a very low uncoupling potency (Fig. 2B and 3), respiration is inhibited at low drug concentrations, which makes it difficult to analyze the relationship between respiratory rate and membrane potential.


Figure 7: Relationship between oxidation rate and membrane potential in the presence of increasing concentrations of ellipticine () and isomers 3 (+), 4 (circle), 5 (), and 6 (bullet). Drug concentrations varied from 0 to 320 nmolbulletmg protein (i.e. 0-160 µM). By comparison the same experiment was carried out with CCCP (Delta; concentration, 0-1 µM). The curves were obtained by simultaneously measuring membrane potential and oxidation rate at pH 7.3 as described under ``Materials and Methods.''




DISCUSSION

Several classes of molecules capable of uncoupling oxidative phosphorylation have been studied. The most typical uncouplers are lipophilic molecules that are weak acids with dissociation constant ranging from 4 to 7(12) . The protonophoric mode of action of these molecules has been widely studied and is summarized in Fig. 8A. Another class of uncouplers is constituted by cations such as cyanine dyes(21) , crystal violet(22) , and cadmium ion(23) . These cations act by modulating the activity of the mitochondrial H/P(i) symporter, and they have no direct protonophoric properties. In fact, their uncoupling-like activity can be observed only in the presence of exogenously added P(i)(21, 22, 23) . Another class of basic, cationic compounds with lipophilic properties, such as local anesthetics, have been found to display an uncoupling activity, even in the absence of P(i)(24) . Their uncoupling properties were initially attributed (24) to a protonophoric mechanism, which is summarized in Fig. 8B. However, Sun and Garlid (25) have proposed a new mechanism by which the protons are driven through the membrane via multimers of the membrane bound drug. More recently, Nagamune et al.(26) have described a cyclic protonophoric mechanism for another lipophilic and basic compound, AU-1421. In the present study, we have investigated the uncoupling activity of lipophilic alkaloids, which behave as weak bases (pK 5.8-7.7). Based on the results of this study, we propose a different mode of action for these molecules.


Figure 8: Model of uncoupling by classical protonophore (A) and protonable ellipticines (B). A, A charged deprotonated and HA uncharged protonated form of uncoupler. B, B uncharged deprotonated and BH charged protonated form of protonable ellipticines. See ``Discussion'' for details.



Ellipticine and its isomers are very potent uncouplers at concentrations ranging between 100 and 300 nmolbulletmg protein, even in the absence of P(i) or lipophilic anion (data not shown). It is important to notice that for concentrations much lower than 100 nmol[chemo]mg protein, all of the possible membrane binding sites for the drug are already saturated (affinity constant 2 times 10^6M, 1 molecule of drug/20 molecules of phospholipid(10) ). Therefore, an increase in ellipticine concentrations enhances the free drug concentration, whereas the concentration of membrane bound drug remains constant. This strongly suggests that the uncoupling activity of ellipticine results from the molecules that are free rather than from those bound to mitochondrial membranes.

According to their chemical structures, the positive charge of the protonated form of ellipticine and its isomers is greatly delocalized. Therefore, it can be predicted that the cationic form of these molecules should be electrophoretically accumulated into the negatively charged mitochondrial matrix. This hypothesis is strongly supported by the depolarization of the membrane potential, which is observed upon addition of ellipticines (Fig. 5).

Both Protonated and Nonprotonated Forms Are Involved in the Uncoupling Properties of Ellipticine, Leading to a Cyclic Protonophoric Mechanism

We have examined the influence of the protonation equilibrium of the drugs on their uncoupling activities by studying the influence of the pH of the medium. We have also compared the activities exhibited by isomers with different pKs at the same pH. These experiments indicated that protonable ellipticines exhibit a maximal uncoupling activity with a pH close to the drug pK. This suggests that the two protonation states of the drugs are involved in the uncoupling mechanism. In fact, the uncoupling efficiency of these molecules is modulated by the transmembrane DeltapH. This suggests that the magnitude of uncoupling depends on the concentration of the neutral form released in the matrix after accumulation of the protonated form. Based on membrane potential measurements, it appears that the protonable molecules (1, 3, 4, 5, 6) induce a depolarization of the mitochondrial inner membrane much more efficiently than the permanent cation ellipticinium does(2) . This comparison suggests that the protonable molecules are able to carry more cationic charges into mitochondria than ellipticinium. Thus, once deprotonated, the neutral form of the molecules can diffuse back from the matrix to the external medium, leading to a cyclic protonophoric mechanism through the mitochondrial membrane (Fig. 8B).

Optimal pH Is Close to the pK of the Drugs

According to the proposed mechanism, the optimal pH for the uncoupling by lipophilic weak bases should be achieved when the proton flux across the membrane is maximum. Optimal conditions should be achieved when equilibria 1, 2, and 3 (Fig. 8B) are shifted toward the formation of the neutral form in the matrix (i.e. when the product resulting from (BH) times (B) is maximum). Optimization of this product leads to pH = pK - (DeltapH/2). Because under physiological conditions the transmembrane DeltapH of rat liver mitochondria does not exceed 0.5 unit (26) , the latter equation may be approximated to pH pK. As it appears in Fig. 3and Fig. 6, experimental results are in good agreement with this approximation. Interestingly, it has been previously observed for derivatives of N-phenyl-2-pyridinamines that the best uncoupling efficiency was found for the molecules with a pK value close to the pH of the incubation mixture(27) .

Conclusion

The results presented in this article provide new evidence concerning the mode of action of ellipticine. In addition to its pharmacological interest, the investigation of the molecular mechanism responsible for the uncoupling properties of this molecule has revealed a new group of uncouplers. From the data reported here on ellipticine and its isomers, it appears that the cyclic mechanism involved in the protonophoric activity of these drugs could be extended to various other weak bases as far as they display a lipophilic character and a pK ranging between 6 and 8.


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.

§
To whom correspondence should be addressed: Biomolécules Conception Isolement Synthèse, CNRS URA 1843, Université Paris-Sud, rue Jean Baptiste Clément, 92296 Châtenay-Malabry, France.

(^1)
The abbreviations used are: CCCP, carbonyl cyanide m-chlorophenylhydrazone; TPP, tetraphenylphosphonium.


ACKNOWLEDGEMENTS

Dr. V. Arondel is acknowledged for critical reading of the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.