(Received for publication, January 24, 1995; and in revised form, June 13, 1995)
From the
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 pH
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.
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
pH. 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.
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.
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) in
the absence (
) or in the presence (
) of 1 µM CCCP or ascorbate
(+N,N,N`,N`-tetramethyl-p-phenylenediamine)
(span cytochrome c-O
) upon the addition of
increasing concentrations of ellipticine (
). B, effect
of increasing concentrations of ellipticine (
), ellipticinium
(
), or isomers 3 (+), 4 (
), 5 (
), or 6 (
)
on oxygen uptake in mitochondria-oxidizing succinate. Experimental
conditions are described under ``Materials and
Methods.''
The effect of ellipticine
isomers and derivative on O uptake was also investigated (Fig. 2B). Except for the quaternarized ellipticinium
derivative(2) , which displays very low stimulation, all
isomers markedly stimulate O
uptake for concentrations
lower than
300 nmol
mg
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
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 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 (
),
and 5 (
) are 244, 68, 230, and 154 µM,
respectively.
Because a positive transmembrane
pH exists in respiring mitochondria, the protonation equilibrium
of the ellipticine isomers may vary as a function of their
localization. Stimulation of O
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
pH. As shown in Fig. 4, when mitochondria are incubated
with ellipticine (90 nmol
mg
protein), the
addition of nigericin clearly reduces the stimulation in O
uptake. This indicates that the presence of a mitochondrial
transmembrane
pH 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.
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 (
), 5 (
), and 6 (
) 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 (
), 5 (
), and 6 (
) 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 (
), 5 (
),
and 6 (
). Drug concentrations varied from 0 to 320 nmol
mg
protein (i.e. 0-160 µM). By comparison the
same experiment was carried out with CCCP (
; 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.''
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
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
(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
(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 nmolmg
protein, even in
the absence of P
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
10
M
, 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).