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J Biol Chem, Vol. 274, Issue 26, 18364-18373, June 25, 1999
From the Tamoxifen has been reported to inhibit
acidification of cytoplasmic organelles in mammalian cells. Here, the
mechanism of this inhibition is investigated using in vitro
assays on isolated organelles and liposomes. Tamoxifen inhibited
ATP-dependent acidification in organelles from a variety of
sources, including isolated microsomes from mammalian cells, vacuoles
from Saccharomyces cerevisiae, and inverted membrane
vesicles from Escherichia coli. Tamoxifen increased the
ATPase activity of the vacuolar proton ATPase but decreased the
membrane potential (Vm) generated by this proton
pump, suggesting that tamoxifen may act by increasing proton permeability. In liposomes, tamoxifen increased the rate of pH dissipation. Studies comparing the effect of tamoxifen on pH gradients using different salt conditions and with other known ionophores suggest
that tamoxifen affects transmembrane pH through two independent mechanisms. First, as a lipophilic weak base, it partitions into acidic
vesicles, resulting in rapid neutralization. Second, it mediates
coupled, electroneutral transport of proton or hydroxide with chloride.
An understanding of the biochemical mechanism(s) for the effects of
tamoxifen that are independent of the estrogen receptor could
contribute to predicting side effects of tamoxifen and in designing
screens to select for estrogen-receptor antagonists without these side effects.
Tamoxifen is the most commonly used treatment for breast cancer
(1). In addition, it is currently being considered for widespread use
in healthy women for breast cancer prevention (2, 3). Yet, despite its
widespread use, its mechanisms of action remain obscure. Tamoxifen is a
known estrogen receptor modulator that acts as an antagonist or partial
agonist. But it has also been reported to have many pleiotropic effects
both in vivo and in vitro that cannot be
explained by an interaction with the estrogen receptor (4). For
example, tamoxifen has been shown to enhance drug sensitivity of
multidrug-resistant cells (5-9), inhibit bone resorption and
osteoporosis both in vivo and in vitro (10), and
inhibit a number of channels, including the volume activated chloride
channel (11, 12) and calcium channels (13-16). These effects have been
attributed to inhibition of P-glycoprotein (17), calmodulin (15), and
direct channel interaction (11), respectively.
Previously, we have observed that tamoxifen inhibits
acidification of intracellular organelles of both estrogen receptor
positive and negative cell lines (18). This inhibition of acidification may be a mechanism for many of the effects of tamoxifen. For example, the effects of tamoxifen on osteoporosis (19), vesicular transport (20,
21), or multidrug resistance (9, 22) are mimicked by blocking the
proton vATPase1 or by a protonophore.
This work addresses the mechanism(s) by which tamoxifen inhibits
ATP-dependent in vitro acidification of
organelles isolated from tissue culture cells, whole tissue, vacuoles
from Saccharomyces cerevisiae, and inverted vesicles
isolated from Escherichia coli. The studies on yeast
vacuolar acidification demonstrate that tamoxifen decreased both
ATP-generated pH gradients and Vm but increased the
ATPase activity of the vATPase. These results suggest that tamoxifen
affects ion permeability of a variety of biological membranes through
interaction with either membrane proteins or the lipid bilayer.
The possibility that tamoxifen acts directly on the lipid bilayer was
addressed with studies of pure lipid vesicles in which tamoxifen
increased the rate of dissipation of the pH gradient. The data suggest
that this occurs by two distinct mechanisms. First, tamoxifen is a
lipophilic weak base with a neutral form that can readily flip-flop
between membranes, and a basic form that is relatively impermeable.
Thus, tamoxifen would accumulate in acidic vesicles, bind protons, and
increase lumenal pH. Importantly, tamoxifen is over 1000-fold more
potent in increasing lumenal pH than the soluble weak base ammonium
chloride. This may be explained by the predominant partitioning of
tamoxifen into the lipid phase, increasing the effective concentration.
However, this mechanism can only be involved in dissipation of a pH
gradient when the lumen is acidic. Second, tamoxifen can mediate
coupled transport of proton or hydroxide with chloride based on the
following observations: 1) it mediates electroneutral dissipation of pH
gradients that is dependent on the presence of chloride or other
halides; 2) it mediates an increased dissipation rate of chloride
gradients; 3) it mediates net proton influx when the external chloride
concentration is greater than the lumenal chloride concentration.
Acidification is crucial for the proper functioning of many cellular
processes, and its disruption may account for many of the pleiotropic
effects described for tamoxifen. The results presented here show that
at low micromolar concentrations, tamoxifen can inhibit acidification
and dissipate pH gradients in a variety of in vitro systems,
supporting in vivo data (18). Whereas this concentration is
higher than required to modulate the estrogen receptor, it is similar
to those reported for many estrogen receptor independent effects.
Importantly, this concentration can readily be achieved in the clinic.
The elucidation of a biochemical mechanism for this estrogen receptor
independent activity of tamoxifen could significantly contribute to the
design of modulators of the estrogen receptor that lack these side effects.
Materials--
Bafilomycin A1, monensin, acridine
orange (AO), pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid),
tamoxifen, Tris-ATP, and nigericin were from Sigma.
BODIPY®-transferrin, lucigenin, and p-xylene-bispyridinium
bromide (DPX) were from Molecular Probes (Eugene, OR). Adriamycin was
from Calbiochem. Concanamycin A was from Fluka (Milwaukee, WI).
Palmitoyloleoyl phosphatidylcholine (POPC) and cholesterol were from
Avanti Polar Lipids (Alabaster, AL).
Acidification of Cellular Microsomes--
Cells were grown in
minimal essential medium supplemented with 10% fetal bovine serum to
confluence in 10-level cell factories (Nunc, Naperville, IL),
trypsinized, washed 3× with cold phosphate-buffered saline, and lysed
with a Dounce homogenizer (pestle A) in 0.25 M sucrose, 20 mM HEPES, pH 7.4, 1 mM DTT, 1 mM
EDTA, and 1× protease inhibitor mix (1 µg/ml leupeptin, 1 µg/ml
pepstatin A, 1 µg/ml aprotinin, and 16 µM
phenylmethylsulfonyl fluoride mixed to 100× before use). The
homogenate was centrifuged twice for 10 min at 3000 × g to remove unbroken cells and nuclei. The supernatant was
layered over 20 ml of 0.5 M sucrose (20 mM
HEPES, pH 7.4, 1 mM DTT, 1 mM EDTA, 1×
protease inhibitor mix) and 1 ml of 2 M sucrose and
centrifuged for 1 h at 100,000 × g (Beckman Ti60 Rotor). Microsomes are collected at the 0.5 and 2 M interface.
To monitor acidification of the total microsomal fraction, the
quenching of AO fluorescence was monitored essentially as described previously (23). Acidic vesicles accumulate AO to high concentrations resulting in the self-quenching of the dye and a decrease of the overall fluorescence. Fluorescence was measured on an SLM Aminco-Bowman series 2 luminescence spectrometer with
Acidification from the recycling endosomal fraction was monitored by
first incubating cells with FITC-transferrin for 30 min before lysis
and isolation of microsomes. Acidification was monitored by excitation
of the FITC fluorophore at 450 and 488 nm and measuring Acidification of Yeast Vacuoles--
Vacuoles from S. cerevisiae were prepared from the protease-deficient strain
BJ2407 (Yeast Genetic Stock Center, University of
California, Berkeley) by sequential flotation through 12 and 8% Ficoll
400 cushion as described previously (24) with the single modification
that 1× protease inhibitor mix and 1 mM DTT was included
in each step. This procedure produced a 25-fold enrichment of the
vacuolar marker Acidification of E. coli Inverted Membrane Vesicles--
InV
were prepared from the DH5 Vm of Yeast Vacuoles--
Oxonol V is a
membrane-permeable anionic fluorescent probe that accumulates into the
inner leaflet of vesicles with positive Vm,
resulting in quenching of fluorescence. Vacuoles were suspended in
chloride or gluconate vesicle buffer with 1 µM oxonol V. Fluorescence was measured with a ATPase Activity of Yeast vATPase--
Vacuoles were diluted in
KCl or potassium gluconate vesicle buffer. Each sample was split into
two, and either 5 µM tamoxifen or carrier was added. Each
of the four resulting samples was again split into two, and either
carrier or 100 nM bafilomycin A1 was added.
Next, 2 mM Tris-ATP was added to each sample, and the
vacuoles were incubated at 30 °C for 15 min. To measure the
phosphate concentration from ATP hydrolysis, an equal volume of
Taussky-Shorr Reagent (1% w/v ammonium molybdate, 2.7% v/v sulfuric
acid, and 5% w/v ferrous sulfate hexahydrate) was added, and the
samples were developed for 15 min. The A660 was
measured (Spectronic Genesys 2) which is linearly related to phosphate
concentration. The bafilomycin-inhibitable ATPase activity was taken as
the difference between the ATPase activity of each condition with or
without 100 nM bafilomycin A1.
Liposome pH--
The lumenal pH (pHL) of liposomes
was assayed with pyranine, a fluorescent dye with a
pKa ~7.3 and a
The pyranine fluorescence was calibrated as a function of pH by
diluting the liposomes with pH 6.2 into a weakly buffered solution of
identical pH (300 mM KCl, 1 mM MES, 1 mM MOPS, 1 mM Tricine, pH 6.2), 1 µM nigericin to allow rapid equilibration with external
pH, and 5 mM DPX to quench external pyranine. The ratio of
the fluorescence emission at
To measure the rate of pH dissipation of liposomes with lumenal pH 6.2, the liposomes were diluted in weakly buffered solution of identical pH
as described above but with no nigericin. Various agents (tamoxifen,
valinomycin, and FCCP) were included as described in the text. The
external pH was shifted to pH 7.3 by addition of 5 mM
glycylglycine, pH 8.4, and the fluorescence ratio was monitored. After
10 min, 1 µM nigericin was added to dissipate the
remaining pH gradient. The pHL was calculated using the
equation pH = x·log(
To assay the effect of addition of NH4Cl or tamoxifen on
liposome pH, liposomes with pHL = 6.2 were diluted into
identical buffer (300 mM KCl, 20 mM MES, 20 mM MOPS, 20 mM Tricine, pH 6.2) containing 5 mM DPX, and the fluorescence ratio was followed after addition of NH4Cl or tamoxifen.
Liposome Chloride Concentration--
Lucigenin is a fluorescent
dye that is collisionally quenched by chloride and other halides but
not by nitrate (26). Lipids dried as described above were rehydrated in
300 mM KNO3, 10 mM K-HEPES, pH 7.3, and 0.5 mM lucigenin. 100 nm unilamellar liposomes were
made, and external dye was removed as described above.
To calibrate the fluorescence of lucigenin as a function of chloride,
the liposomes were diluted in buffer (300 mM
KNO3, 10 mM K-HEPES, pH 7.3) with 1 µM tributyltin (TBT) a Cl
To measure the chloride permeability, the fluorescence was followed in
liposomes after the addition of 50 mM KCl. After 10 min, 1 µM TBT and 1 µM nigericin were added. The
chloride concentration was calculated using the Stern-Volmer equation
with k calculated from the titration curve.
Octanol Partitioning of Tamoxifen--
The concentration of
tamoxifen was measured by its absorbance peak at 245 nm. The
A245 of 20 µM tamoxifen in
phosphate-buffered saline, HCl, pH 1, or KOH, pH 13, solution was
acquired. Then, 1 µl of octanol was added, the solution was vortexed,
and the A245 of the aqueous phase was acquired.
In Vitro Acidification of Vesicles from Mammalian Cells
Total Microsomal Preparation--
The mechanism by which tamoxifen
inhibited acidification of intracellular organelles was first addressed
by testing whether tamoxifen acted directly on the organelles or
indirectly through soluble modulators. Acidification of organelles was
assayed in vitro using microsomes isolated from MCF-7/ADR
cells that are free of detectable soluble cytosolic proteins.
Acridine orange (AO) was used as a probe for lumenal acidification
(23). As vesicles acidify, they accumulate AO to self-quenching concentrations and deplete the extravesicular free AO, resulting in a
decrease in total fluorescence. Acidification was initiated by the
addition of ATP to a purified microsomal fraction in the absence of
cytosol (Fig. 1A, at
t = 300 s). Over the subsequent 1200 s, there
was a reduction of the AO fluorescence, suggesting an accumulation of
AO within the lumen of the microsomes. Nigericin, a
K+/H+ exchanger that rapidly dissipates pH
gradients, was added at the end of each reaction (t = 1500 s). In all experiments the AO fluorescence returned to its
pre-ATP levels. This indicates that the decreased fluorescence was the
consequence of the generation of a pH gradient.
When MCF-7/ADR vesicles were pretreated with tamoxifen for 30 min,
there was a dose-dependent inhibition of AO quenching (Fig. 1A). Inhibition was evident when using 1 µM
tamoxifen, and acidification was totally blocked with 8 µM tamoxifen. To quantify the effects of tamoxifen on
acidification, we plotted acidification (as assayed by quenching of AO
fluorescence) as a function of tamoxifen concentration (Fig. 1A,
inset). The ID50 for maximal quenching is
approximately 3 µM, which is in the same range that
tamoxifen inhibits acidification in vivo (18). As a positive
control, we employed bafilomycin A1, a potent and specific
inhibitor of the vATPase responsible for acidification of all
intracellular compartments (27).
To determine the time course for the inhibition of acidification by
tamoxifen, the drug was added 10 min after addition of ATP (Fig.
1B). Addition of tamoxifen rapidly reversed acidification and caused an almost complete dissipation of the pH gradient within 5 min. Addition of bafilomycin A1 dissipated the pH gradient
at a much slower rate, even when used at 100 nM, which is
10 times the concentration that blocked 95% of acidification (see Fig. 1A). Addition of nigericin (Fig. 1B) and monensin
(data not shown) dissipated the pH gradient significantly faster than
tamoxifen. Thus, the time course of alkalinization by tamoxifen is
distinguishable from both rapidly acting electroneutral protonophores
and inhibitors of the vATPase.
The fact that the in vitro acidification assay used purified
microsomes in the absence of cytosolic or nuclear components indicates
that the effects of tamoxifen on pH should be independent of cytosolic
factors, such as the estrogen receptor, and of both transcription and
translation. In addition, tamoxifen had similar effects on in
vitro acidification of microsomes isolated from liver and kidney
tissue from mice (data not shown). Therefore, the effect of tamoxifen
on organelle acidification appears to be a general phenomenon.
In Vitro Acidification of Recycling Endosomes--
To specifically
examine the acidification of the endosomes in vitro, we
assayed in vitro acidification using FITC-transferrin which
is constrained by the transferrin receptor to the endocytic pathway.
MCF-7/ADR cells were incubated with FITC-transferrin before lysis and
isolation of microsomes. Since this assay is not based on the
redistribution of probes, it further serves as verification that AO
quenching resulted from vesicular acidification. Upon addition of ATP,
there was a decrease in the ratio of the FITC emission (Fig.
1C). This signal was judged to be the consequence of
acidification since it was reversed upon the addition of nigericin. The
addition of 2.5 µM tamoxifen partially reversed the
acidification in these organelles. This was further reduced by raising
the tamoxifen concentration an additional 2.5 µM. This
indicates that the recycling endosomes were one of the compartments in
this in vitro assay whose acidification was blocked by tamoxifen.
Mechanisms of Organelle Acidification
Acidification of intracellular organelles utilizes an electrogenic
proton pump (the vATPase) and chloride channels (28, 29). The vATPase
couples ATP hydrolysis to proton movement. The unidirectional movement
of the proton generates an inside positive Vm which
limits acidification. The chloride channels allow passive chloride
influx into the organelles, dissipating the Vm.
Tamoxifen could inhibit acidification by the following possible
mechanisms: direct inhibition of the vATPase; indirect inhibition of
the vATPase through modulation of the Vm (such as
blocking a chloride conductance); inhibition of acidification by a weak
base effect or dissipation of pH gradients as a protonophore. There
exists evidence in support of each of these mechanisms.
Inhibition of the vATPase--
Tamoxifen has been reported to
inhibit acid secretion by avian osteoclasts through inhibition of the
plasma membrane vATPase activity. This activity has been attributed to
the antagonism by tamoxifen of the membrane-bound
calmodulin-dependent cyclic nucleotide phosphodiesterase,
which regulates the vATPase (30).
Inhibition of the Chloride Channel--
Tamoxifen has been
reported to inhibit the volume-activated chloride channel (11).
Dissipation of pH Gradient by a Weak Base Effect--
A weak base
(such as ammonium chloride) will rapidly cross the membrane in a
neutral (i.e. NH3) form and bind protons in the interior causing an alkaline shift. The charged form of these molecules
will accumulate according to the Henderson-Hasselbach equilibrium.
Tamoxifen is a weak base with a pKa of 6.9 when
measured by NMR in 10% Triton solution (31). At a free tamoxifen
concentration of 8 µM, a pH 7.3-5.3 gradient will result in <200 µM lumenal concentration. This is less than the
buffering capacity of the organelles, and this should not significantly perturb lumenal pH. Thus, we initially considered this mechanism unlikely.
Dissipation of pH Gradient by Increasing Proton
Permeability--
Tamoxifen partitions into lipids, increases membrane
fluidity, and decreases lipid peroxidation (32). If the charged
protonated form of tamoxifen were membrane-permeable, tamoxifen would
act like a classic protonophore. This mechanism has been proposed for
the ability of many amine local anesthetics to uncouple respiration (33, 34).
Each of these potential mechanisms has distinct consequences for ATPase
activity and Vm of the acidic organelle (Table I). If tamoxifen inhibits the vATPase, it
would decrease the ATPase activity. In addition, it should decrease
Vm of the organelles since the proton pumping is
generating the Vm. If tamoxifen inhibits the
chloride channel, it would increase Vm, since the
chloride channel serves to dissipate Vm. As a
consequence of the increased Vm, the vATPase cannot pump protons, resulting in a decreased rate of ATP hydrolysis. If
tamoxifen is a protonophore, it should decrease Vm by allowing protons to permeate and increase ATPase activity by decreasing the electrochemical gradient against which the vATPase must
pump. A weak base should slightly increase Vm and ATPase activity since it dissipates the proton gradient in favor of an
electrical gradient.
The predictions of these mechanisms were tested on isolated vacuoles
from S. cerevisiae. Vacuoles from S. cerevisiae
offer several advantages in biochemical studies of the actions of
tamoxifen as follows. 1) They further address the specificity of the
effects of tamoxifen (S. cerevisiae are known not to have an
estrogen receptor). 2) They use the same basic machinery as mammalian
organelles, a vATPase and chloride channel, to generate the proton
gradient. 3) They can be purified in large amounts. It is very
difficult to prepare mammalian organelles to the high purity required
to assay Vm and ATPase activity. In yeast vacuole
preparations, the vATPase represents ~50% of all ATPase activity,
which is much higher than attainable for endosome or Golgi preparations.
In Vitro Acidification of Yeast Vacuoles and E. coli Membrane
Vesicles
Acidification of Yeast Vacuoles--
To test if tamoxifen
inhibited acidification in yeast vacuoles, in vitro
acidification of vacuoles was assayed using AO. The buffer was
pre-equilibrated with either carrier (Me2SO), tamoxifen (2 µM or 8 µM), ammonium chloride (1 mM), or concanamycin A (10 nM), or in potassium
glutamate instead of KCl buffer (Fig.
2A). ATP (1.5 mM)
was added at 50 s to initiate acidification, and at 400 s
nigericin (1 µM) was added to dissipate pH gradients. As
observed in mammalian microsomes, tamoxifen shows a
dose-dependent inhibition of acidification, with complete
inhibition at 8 µM. This strongly implies that tamoxifen
inhibits acidification independent of the estrogen receptor which is
not found in yeast. Acidification was slightly inhibited by the weak
base ammonium chloride (1 mM). This is 1000-fold greater
than the concentration of tamoxifen required to achieve similar
inhibition.
Addition of tamoxifen to pre-acidified vacuoles (at 250 s in Fig.
2B) resulted in a rapid alkaline step followed by slower alkalinization. The step is reminiscent of a weak base which rapidly establishes equilibrium across vesicles. Thus, a comparison was made of
the effects of adding tamoxifen and the weak base ammonium to
pre-acidified vacuoles (Fig. 2B). Addition of ammonium (1 mM) indeed caused a step alkalization. However, the
vacuoles slowly re-acidify after addition of ammonium but continue to
alkalinize after addition of tamoxifen.
Membrane Potential--
The fluorescent dye Oxonal V was used to
monitor Vm. Oxonol V contains two delocalized
negative charges and is highly lipophilic. In the presence of vesicles
with positive Vm, it accumulates in the lumen and
inner leaflet, resulting in fluorescence quenching (35). Unlike AO,
which exhibits quenched fluorescence in the presence of acidified
vesicles regardless of the number of non-acidified vesicles present,
Oxonol V will report an average Vm for all vesicles.
This necessitates the use of pure preparations, such as the yeast vacuoles.
The Vm was monitored in either KCl or potassium
gluconate buffer (Fig. 3A). At
50 s, tamoxifen (10 µM) or carrier was added, and
acidification was initiated by adding ATP (2.5 mM) at
200 s. A larger positive Vm was generated in
potassium gluconate than in KCl which implicates a chloride
permeability in dissipating the Vm of the vacuoles.
Tamoxifen significantly decreased the Vm generated
by the vATPase. This suggests that tamoxifen may increase ion
permeability thus decreasing the Vm.
ATPase Activity--
To assay specifically the vATPase activity,
the effects of tamoxifen were quantified on the bafilomycin-inhibitable
ATPase activity. Replacing chloride with gluconate decreased the
bafilomycin-inhibitable ATPase activity (Fig. 3B), further
confirming that chloride provided the counter-ion transport to
dissipate the Vm. Addition of tamoxifen caused an
increase of ATPase activity in both conditions, with a more dramatic
increase in gluconate buffer (Fig. 3B).
In summary, in yeast vacuoles tamoxifen inhibited
ATP-dependent acidification, decreased
Vm, and increased bafilomycin-inhibitable ATPase
activity. These observations are consistent with the hypothesis that
tamoxifen increases membrane permeability to protons, either through
direct lipid interaction or through proteins or modulators (Table
I).
Acidification of E. coli Inverted Membrane Vesicles--
To test
the protein and lipid specificity of acidification inhibition by
tamoxifen, the effects of tamoxifen on ATP-dependent acidification in E. coli inverted vesicles (InV) was
assayed. Unlike mammalian or yeast vesicles, InV utilize the
F0F1-ATPase for acidification and are composed
of different types of lipids, including an abundance of cardiolipin and
a lack of sterols.
As shown in Fig. 4, the presence of
tamoxifen inhibited acidification in InV with a similar dose dependence
as observed in mammalian and yeast vesicles (Figs. 1A and
2A). Similarly, addition of tamoxifen to E. coli
vesicles pre-acidified with ATP resulted in similar rates of
alkalinization (data not shown) as mammalian and yeast vesicles (Figs.
1A and 2A). These observations indicate that
tamoxifen can dissipate pH gradients across a diverse spectrum of
native biological membranes.
Liposomes
pH Gradients--
The effect of tamoxifen on pH gradients was
tested in pure lipid vesicles. This system was used both because the
effects of tamoxifen on acidification were observed in diverse
biological membranes and because the results on the ATPase activity
were consistent with tamoxifen affecting membrane permeability to protons.
Liposomes were loaded with pyranine, a hydrophilic, non-permeable
fluorescent pH indicator to assay proton permeability. The log of the
ratio of the fluorescence emission of pyranine when excited at
To mimic the acidified lumen of organelles, liposomes were made with
the pHL buffered at 6.2. The pHL was monitored
while the external pH (at 50 s) was shifted to 7.3 (Fig.
6A) in the presence tamoxifen
(0, 0.5, or 2 µM). Nigericin (1 µM) was
added at 700 s to dissipate pH gradients. In the absence of
tamoxifen, the pHL increased less than 0.2 pH units over
the 10-min span. In the presence of tamoxifen, after a shift of
external pH, there was a rapid step increase in pHL. The
proton permeability of liposomes after the step increase is difficult
to compare with the control, since the pH gradient has decreased.
Tamoxifen did not induce detectable leakage of pyranine, and in
solution, tamoxifen does not affect pyranine fluorescence (data not
shown).
The effects of tamoxifen on pHL were contrasted with the
effects of other pH perturbants with known mechanisms of action, specifically a protonophore (FCCP), a potassium ionophore
(valinomycin), and a weak base (NH4Cl) (Fig.
6B). FCCP at saturating concentrations only slowly
dissipated the pH gradient. This is because FCCP allows free movement
of only protons. Thus proton efflux down its gradient generates a
Vm preventing further proton movement. The presence
of valinomycin, a K+-selective ionophore which would
dissipate Vm, caused a faster dissipation of the pH
gradient than FCCP (Fig. 6C). Here, protons can efflux down
the concentration gradient without generation of Vm.
Thus, these liposomes were more permeable to protons than potassium. As
expected, the combination of FCCP and valinomycin immediately
dissipated the pH gradient (data not shown) comparable with the effects
of nigericin. The weak base NH4Cl caused a step alkaline
shift, followed by a slow alkaline drift. Here, the alkaline shift is
caused by the selective diffusion of the basic NH3 into the
vesicles, whereas the acidic NH4+ is
impermeable. Of the three agents tested, the effect of micromolar concentrations of tamoxifen is most similar to the effect observed at
millimolar concentrations of NH4Cl; the step alkalinization upon changing the external pH was similar, but the subsequent dissipation of pH was faster with 0.5 µM tamoxifen (Fig.
6A).
The potential contribution of a weak base effect in the mechanism of
tamoxifen action was further explored using liposomes in the absence of
a pre-existing pH gradient. The pHL was monitored and
tamoxifen (2 and 8 µM) or NH4Cl (5 mM) was added at 50 s. At 700 s, nigericin was
added to dissipate the pH gradient. As expected, upon addition of the
weak base NH4Cl, the non-protonated species
(NH3) rapidly diffused into the liposomes, where it was protonated causing alkalinization of the lumen (Fig.
7A). The pH gradient slowly
dissipated by either leakage of H+ or
NH4+. Similarly, addition of tamoxifen
caused alkalinization of liposomes, followed by more rapid pH
equilibration (Fig. 7A). This suggests that like ammonia,
tamoxifen-free base rapidly enters liposomes, causing a step alkaline
shift in the lumen while the protonated tamoxifen is less
permeable.
The observation that tamoxifen exerted similar effects to
NH4Cl at 3 orders of magnitude lower concentration suggests
that it may be highly concentrated within liposomes. The extent of lipid partitioning of tamoxifen was examined by measuring the partitioning coefficient of tamoxifen between octanol and aqueous buffer. Tamoxifen in aqueous solution was equilibrated with either 1:1000 or 1:100 volume of octanol, and the concentration of tamoxifen left in the aqueous phase was measured by absorbance (Fig.
7B). Notice that 1:1000 volume octanol was able to extract
approximately 50% of tamoxifen in aqueous solution, suggesting that
tamoxifen partitions 3 orders of magnitude greater into the lipid
phase. Octanol partitioning was repeated with the aqueous phase
buffered to pH 1 and pH 13 to examine possible differences in
partitioning between the charged and neutral forms of tamoxifen,
respectively. The same result was obtained at both pH values (data not
shown). This is consistent with the difference in concentrations of
NH4Cl and tamoxifen required for the same quantitative
effect. It may also contribute to the observation that pyranine reports
a lower pHL in the presence of tamoxifen even after
addition of nigericin (Fig. 7A). Tamoxifen could effectively
give the liposomes a positive surface charge, which has been shown to
effect pyranine fluorescence (36).
In both yeast vacuoles (Fig. 2B) and liposomes (Fig.
7A), addition of tamoxifen or ammonium caused similar pH
jumps, but tamoxifen subsequently caused a more rapid equilibration of
pH. This suggests that other mechanism(s) may contribute in addition to
the weak base effect. In liposomes with an acidic lumen, the initial
weak base alkaline jump is too large to allow an assessment of the rate
of subsequent pH dissipation. Therefore, we tested the effects of
tamoxifen and a weak base on liposomes with an alkaline interior pH of
8.1. The pHL was monitored after shifting the external pH to 6.9 by the addition of 4 mM MES, pH 5, at 50 s
(Fig. 8A). The pH gradient was
dissipated more quickly with increasing concentrations of tamoxifen. In
contrast, the effect of NH4Cl (10 mM) was
indistinguishable from the control. Thus, the dissipation of pH by
tamoxifen cannot be solely explained as a weak base effect.
To explore potential ionophoretic mechanisms, tamoxifen was compared
with FCCP and valinomycin (Fig. 8B). Addition of FCCP did
not substantially increase the rate by which the pH gradient was
dissipated, presumably because proton leakage is limited by the
Vm. This is substantiated by the observation that valinomycin caused a greater dissipation of the pH gradient than FCCP.
The observation that tamoxifen diminished the pH gradient faster than a
saturating concentration of FCCP (Fig. 8B) implies that
tamoxifen cannot be a pure protonophore. Any pure protonophore will,
like FCCP, allow free proton movement but be limited by Vm. Importantly, when both tamoxifen and valinomycin were included, the effect was additive and not synergistic.
This implies that tamoxifen mediated proton movement is
electroneutral. If tamoxifen mediated an electrogenic
process (e.g. pure protonophore), the dissipation of the
Vm by valinomycin would dramatically increase the
effect of tamoxifen. For example, the presence of valinomycin allows
FCCP to immediately dissipate any pH gradient.
Chloride Permeability--
If tamoxifen mediates bi-directional
electroneutral transport of protons, then a second ion must be
co-transported. We first asked if this ion could be chloride. We
examined the effect of tamoxifen on the influx of chloride into
liposomes. Lucigenin, a fluorescent dye that is collisionally quenched
by chloride, was employed (39). Liposomes were loaded with
KNO3 buffer and lucigenin. The lumenal chloride
concentration can be accurately calibrated by the fluorescence (data
not shown).
Lumenal chloride concentration in the liposomes was monitored after
addition of 50 mM KCl to the external solution (Fig.
9A). Tamoxifen caused a
dose-dependent increase in the rate of chloride influx.
Since chloride is more membrane-permeable than potassium, unidirectional chloride movement is also expected to be limited by
Vm (40). Indeed, the presence of valinomycin
increased the rate of chloride equilibration. To test if tamoxifen was
affecting chloride permeability solely by dissipation of the
Vm, tamoxifen was added to liposomes in the presence
of a concentration of valinomycin (1 µM) that completely
dissipates the Vm (Fig. 9A). The addition
of 4 µM tamoxifen increased the rate of chloride influx
observed in the presence of valinomycin. This suggests that tamoxifen
must be having an effect on chloride permeability independent of any
effects on Vm. In addition, the fact that the
effects tamoxifen and valinomycin were additive on chloride permeability also suggests that the chloride transport is
electroneutral.
Since tamoxifen seems to increase both proton and chloride
permeability, we next addressed the question of whether tamoxifen mediates their coupled transport (i.e. the crossing of HCl
but not H+ or Cl
To further test if tamoxifen is acting as a coupled
Cl Tamoxifen inhibits ATP-dependent acidification in
intact cells (18), mammalian organelles (Fig. 1), yeast vacuoles (Fig. 2), and InV (Fig. 4). It also dissipates pH gradients in liposomes (Figs. 6-9). The tamoxifen-dependent dissipation of the pH
gradient is independent of all proteins including the estrogen receptor.
Our results suggest that tamoxifen affects transmembrane pH through at
least two independent mechanisms as follows: as a weak base and as a
mediator of coupled transport of proton/hydroxide and chloride. For
vesicles with an acidified lumen, tamoxifen causes a rapid alkaline
shift of the pHL which is most likely a weak base effect
(Fig. 10). We propose tamoxifen is
highly concentrated within the leaflets of membranes. Since tamoxifen
is a weak base, its neutral form can readily flip between inner and
outer leaflets while the charged form flips much less readily.
Therefore, it will accumulate within the inner leaflet of acidic
organelles, causing a step alkalinization.
A Mechanism for Tamoxifen-mediated Inhibition of
Acidification*
§,
, and**
Laboratory of Cellular Biophysics,
Rockefeller University, New York, New York 10021 and the
¶ Department of Biochemistry, Michigan State University,
East Lansing, Michigan 48824
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ex = 488 nm and
em = 530 nm. Microsomes (80 µg of protein) were
suspended in 2.5 ml of vesicle buffer (125 mM KCl, 5 mM MgCl2, 20 mM HEPES, pH 7.4, 1 mM DTT, 1 mM EDTA, 2 mM
NaN3), with 6 µM AO (5 mM stock
in H2O) in a cuvette. To examine the ability of vesicles to
generate a 
pH in the presence of tamoxifen or bafilomycin
A1, 0, 1, 2, 4, or 8 µM tamoxifen (10 mM stock in EtOH) or 10 nM bafilomycin
A1 (10 mM stock in 10% Me2SO) was
added. After equilibration for 30 min at 25 °C, 1 mM
Tris-ATP was added to begin acidification (100 mM stock,
titrated to pH 7.4 with 1 M Tris base before use). Twenty
minutes later, 2.5 µM nigericin (10 mM stock
in EtOH) was added to dissipate any pH formation. To study the
effects of tamoxifen and bafilomycin A1 on vesicles with a
pre-existing pH tamoxifen or bafilomycin A1 were added
10 min after the addition of Tris-ATP.
em = 520 nm as described previously (18).
-mannosidase (data not shown). Acidification was
measured using AO as described above. For acidification in chloride-free solution, gluconate or glutamate was used in vesicle buffer instead of chloride.
strain as described (25).
Acidification was measured using AO as described above.
ex = 600 nm and em = 630 nm. After fluorescence had equilibrated,
vacuoles were added, and the fluorescence was allowed to
re-equilibrate. Then, 1 mM Tris-ATP was added, and the
resulting positive Vm was manifested in fluorescence quenching.
ex = 405 nm in its
acid form (
3 charge) and a ex = 455 nm in its basic
form (4 charge). To prepare pyranine-loaded liposomes, lipids (2 mg of POPC, 1 mg of cholesterol) supplied in chloroform suspension were
dried in a round bottom flask under argon for 2 h. The lipids were
then resuspended in acidic or alkaline liposome buffer (300 mM KCl, 20 mM MES, 20 mM MOPS, 20 mM Tricine titrated with KOH to either pH 6.2 or pH 8.1)
containing 0.5 mM pyranine. The suspension was incubated at
room temperature overnight and then freeze-thawed 6 times. Unilamellar
liposomes were prepared by extrusion 3 times through two stacked 100-nm
Nucleopore (Corning/Costar Scientific, Acton, MA) polycarbonate filters
in an Avestin (Vancouver, British Columbia, Canada) extruder at 600 pounds/square inch. More than 95% of external pyranine was separated
from the liposomes by sequentially running through NAP-10 and NAP-25
desalting columns (Amersham Pharmacia Biotech). Internal pyranine
leakage was <1% per day, and liposomes were used within 1 week of preparation.
em = 510 nm was monitored with dual excitation wavelengths of ex = 405 and
ex = 455 nm. Sequential aliquots of 0.1 mM
glycylglycine, pH 8.4, were added to increase pH (see Fig. 5). The
fluorescence was measured after each addition, and the pH was measured
using a pH meter. The logarithm of the fluorescence ratio was linearly
dependent on the pH. The curve generated by a least squares fit between
pH 6.2 and 7.9 resulted in 
2 >0.99. The calibration
curve for the liposomes of lumenal pH 8.1 was generated identically
except sequential aliquots of 0.1 mM K-MES, pH 5.0, were
added for titration, and the curve was generated between pH 8.1 and pH
6.4.
ex = 405 nm/ex = 405 nm) + c, where x and
c are constants from the least square fit of the titration
curve. To measure the rate of pH dissipation of liposomes with
pHL = 8.1, the identical procedure was followed except 5 mM K-MES, pH 5.0, was added to shift the external pH to
6.9.
-OH
exchanger, and 1 µM nigericin, a
K+-H+ exchanger. This results in rapid net
dissipation of KCl gradient. Aliquots of 0.5, 1, 2, 4, 8, 16, and 32 mM KCl were added, and lucigenin fluorescence
(ex = 370 nm/ex = 505 nm) was recorded. The fluorescence was fitted to the Stern-Volmer equation:
F0/F = 1 + k[Cl],
where F0 is the fluorescence in the absence of chloride.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of tamoxifen on in vitro
acidification of MCF-7/ADR organelles. A,
preincubation with tamoxifen. Acridine orange is a weak base that
accumulates to self-quenching concentrations into acidic compartments.
Thus, the presence of acidic compartments decreases the total
fluorescence by decreasing the concentration of free AO outside those
compartments. Microsomes were suspended in AO, and after establishing
base line, 1 mM Tris-ATP was added to begin acidification
(at 300 s). This caused a slow decrease of total fluorescence over
1200 s (Ctrl). Addition of the protonophore nigericin
(5 µM Nig) at t = 1500 returned the fluorescence levels demonstrating the fluorescence
decrease was the consequence of acidification. This inhibitory effect
of tamoxifen (Tam) on acidification was apparent at 1 µM and increased in a dose-dependent manner
(2, 4, and 8 µM). Pretreatment of microsomes with 10 nM bafilomycin A1 (Baf) also blocked
acidification. Inset, dose-response of tamoxifen on
acidification. Acidification was assayed as in A.
B, tamoxifen added during the acidification. Ten minutes
after the addition of 1 mM Tris-ATP, 8 µM
tamoxifen or 100 nM bafilomycin A1 was added
which rapidly reversed acidification of the organelles. In the absence
of tamoxifen or bafilomycin A1, the organelles continued to
acidify. Ten minutes later 5 µM nigericin was added.
C, acidification in recycling endosomes assayed by
FITC-transferrin. Cells were incubated with FITC-transferrin, which is
endocytosed and localized within the endosomes. After lysing the cells
a microsomal fraction was harvested. The fluorescence emission at 520 nm was monitored in response to excitation at 488 and 450 nm, and the
ratio was plotted. When excited at 488 nm, the fluorescence of FITC
increases with increasing pH, but when excited at 450 nm, the
fluorescence of FITC is pH-independent. Therefore, a decreasing ratio
indicates acidification. Upon addition of ATP (t = 1080 s) there was acidification of the lumen of the microsomes as
assayed by a decrease in the ratio of the 488:450 nm emission.
Nigericin was added (t = 2500 s) to confirm that
the fluorescent shift was the result of acidification. Successive
additions of 2.5 µM tamoxifen caused alkalinization of
endosomes. Nigericin was added at the end to equilibrate pH.
Predicted effects of potential mechanisms of tamoxifen on
Vm and ATPase activity
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Fig. 2.
Effect of tamoxifen on acidification of yeast
vacuoles. A, effect of tamoxifen, ammonium sulfate, and
chloride on yeast vacuole acidification. Vacuoles were suspended in
buffer (KCl except potassium glutamate labeled) containing AO in the
presence of tamoxifen (2 or 8 µM Tam),
ammonium sulfate (1 mM
NH4+), or potassium glutamate instead of
KCl. ATP (1.5 mM) was added at 50 s to initiate
acidification and nigericin (1 µM Nig) was
added at 400 s. The presence of tamoxifen caused a
dose-dependent inhibition of acidification. Ammonium
sulfate, at much higher concentrations, also caused a slight
inhibition. When glutamate was used instead of chloride, acidification
decreased, consistent with a chloride channel dissipating the
Vm. B, effect of adding tamoxifen or
ammonium sulfate during acidification of yeast vacuoles. Vacuoles were
in a KCl buffer containing AO, and acidification was initiated by
addition of ATP (1.5 mM) at 50 s. At 250 s,
either ammonium sulfate (1 mM) or tamoxifen (2 µM) was added. Each caused a step increase in AO
fluorescence, indicating alkalinization. Subsequently, AO fluorescence
continued to increase when tamoxifen was added but slowly decreased
when ammonium sulfate was added.
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Fig. 3.
Effect of tamoxifen on Vm
and vacuolar ATPase activity of yeast vacuoles.
A, effect of tamoxifen (Tam) and chloride on
Vm of yeast vacuoles. Vacuoles were suspended in KCl
or potassium gluconate (Kgluc) buffer with 1 µM Oxonol V (OxV). Oxonol V is a
membrane-permeable anionic dye. Thus, it accumulates into vesicles of
positive Vm, resulting in fluorescence quenching.
Addition of ATP resulted in a vATPase generated inside-positive
Vm of the vacuoles and quenching of Oxonol V
fluorescence. In the absence of tamoxifen, the Vm
was greater in potassium gluconate than KCl, consistent with a chloride
channel dissipating the Vm. The presence of
tamoxifen decreased the ATP-generated Vm in both KCl
and potassium gluconate to similar levels. B, effect of
tamoxifen and chloride on bafilomycin-inhibitable ATPase activity of
yeast vacuoles. ATPase levels were quantified by measuring released
phosphate as described under "Experimental Procedures." Samples
were split in two and processed ± bafilomycin A1 to determine
vATPase activity. In the absence of tamoxifen, ATPase activity was
greater in chloride than gluconate. This is because in the absence of
chloride, there is a large Vm against the vATPase.
Tamoxifen increased the ATPase activity in both buffers.
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Fig. 4.
Effect of tamoxifen on acidification of
E. coli inverted membrane vesicles. InV were
suspended in KCl buffer with 6 µM AO in the presence
(0.63, 2, and 10 µM) or absence of tamoxifen
(Tam). ATP (1.5 mM) and nigericin
(Nig) (1 µM) were added at 100 and 400 s.
Similar to both mammalian microsomes (Figs. 1 and 2) and yeast vacuoles
(Fig. 3), tamoxifen inhibited ATP-dependent
acidification.
ex = 405 nm and ex = 455 nm is linearly
dependent on the pH (Fig. 5) (36, 37).
Two steps were taken to ensure that only pHL was measured
and to permit the discrimination between dissipation of pH gradient and
the lysis or dye leakage from liposomes. First, greater than 95% of
external pyranine was removed by gel filtration. Second, the
membrane-impermeable quencher DPX was added to the external solution
(38) which effectively quenched all remaining non-lumenal pyranine
fluorescence.
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Fig. 5.
Titration curve of pyranine-loaded
liposomes. Pyranine is a non-membrane-permeable ratiometric pH
indicator. Its acidic form is fluorescent when excited at 405 nm, and
its basic form is fluorescent when excited at 455 nm. Liposomes
(pHin 6.3) were loaded with pyranine and diluted into
buffer with pHout 6.3 in the presence of nigericin, to
allow rapid equilibration between internal and external pH. The raw
fluorescence when excited at 405 and 455 (left axis) as well
as the ratio (right axis, log scale) were monitored. DPX, a
non-permeable quencher of pyranine fluorescence, was added and
decreased the raw fluorescence by only the dilution factor, indicating
that virtually all pyranine fluorescence is from the liposome lumen.
Aliquots of MES were added to decrease the external pH and internal pH
(due to the presence of nigericin), and the pH was measured using a pH
meter. There was no decrease in total fluorescence throughout the
experiment, indicating no dye leakage or DPX influx.
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Fig. 6.
Rate of pH equilibration of pH 6.3 liposomes
in a pH 7.3 bath. Pyranine-loaded liposomes with pHin
6.3 was diluted into KCl buffer of same pH and 10 mM DPX
and one of the follow compounds: carrier, tamoxifen (Tam)
(0.5 and 2 µM), NH4Cl (1 mM),
FCCP (1 µM), or valinomycin (Val) (1 µM). At 20 s, 5 mM potassium
glycylglycine, pH 8.4, was added to raise pHout to 7.3. At
700 s, 1 µM nigericin (Nig) was added to
equilibrate pH. A, with the presence of tamoxifen, the pH
shift caused a rapid alkaline shift of the lumen. This cannot be due to
lysis because there was no decrease of total fluorescence indicating
lack of dye leakage into DPX-containing external buffer. Ammonium, at
2000× concentration, caused a similar alkaline jump. But the rate of
pH dissipation was faster after the alkaline jump with tamoxifen than
ammonium. B, FCCP and valinomycin each increased the rate of
pH dissipation, but did not cause an alkaline jump upon change of
external pH.
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Fig. 7.
Basis for weak base effect of tamoxifen.
A, effect on liposome pH by weak base addition.
Pyranine-loaded liposomes with pHin 6.3 were diluted into
KCl buffer of same pH and 10 mM DPX. At 50 s,
tamoxifen (Tam) (2 and 8 µM) or
NH4Cl (5 mM) was added. At 700 s,
nigericin (Nig) (1 µM) was added. Addition of
tamoxifen or NH4Cl resulted in alkalization of the lumen,
presumably due to selective influx and protonation of the uncharged
species. Importantly, following the alkaline jump, the pH
re-equilibrated much faster when tamoxifen was used, suggesting a
tamoxifen-mediated proton permeability. B, octanol
partitioning of tamoxifen. Tamoxifen (20 µM) in
phosphate-buffered saline was mixed with 1:1000 or 1:100 volume of
octanol. The tamoxifen concentration of the aqueous phase was
determined using absorbance spectroscopy. Notice the 1:1000 volume
octanol was able to extract approximately ~50% tamoxifen from the
aqueous phase. This suggests that tamoxifen partitions 3 orders of
magnitude into the lipid phase and is consistent with the potency of
tamoxifen compared with ammonium in A.
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Fig. 8.
Rate of pH equilibration of pH 8.1 liposomes
in a pH 6.9 bath. Pyranine-loaded liposomes with pHin
8.1 was diluted into KCl buffer of the same pH and 10 mM
DPX and one of the following compounds: carrier, tamoxifen
(Tam) (0.5, 2, and 8 µM), NH4Cl
(10 mM), FCCP (1 µM), valinomycin
(Val) (1 µM), or both tamoxifen (8 µM) and valinomycin (1 µM). At 50 s, 5 mM K-MES was added to lower pHout to 6.9. A, increasing concentrations of tamoxifen caused a
dose-dependent increase of pH equilibration. The weak base
NH4Cl, at 10× concentration used to observe a significant
weak base effect (Fig. 6), had no effect. B, FCCP and
valinomycin each increased the rate of pH dissipation. Tamoxifen
increased the rate faster than saturating concentrations of FCCP.
Tamoxifen and valinomycin together were additive and not synergistic.
These two observations suggest that tamoxifen cannot be a pure
protonophore but must mediate an electroneutral process.
Nig, nigericin.
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Fig. 9.
Effect of chloride on tamoxifen-mediated
proton permeability. A, rate of chloride influx into
liposomes. Lucigenin, a non-permeable fluorescent probe that is
collisionally quenched by chloride, was used to assay liposome chloride
concentration. Lucigenin-loaded liposomes made with an internal
solution of 150 mM KNO3 were diluted into 150 mM KNO3 buffer and one of the follow compounds:
carrier, tamoxifen (Tam) (2, 4, and 8 µM),
valinomycin (Val) (1 µM), or both tamoxifen (4 µM) and valinomycin (1 µM). At 50 s,
50 mM KCl was added, and the internal chloride
concentration was followed using lucigenin fluorescence. Tamoxifen
caused a dose-dependent chloride influx. Valinomycin also increased
chloride influx by dissipating Vm. Importantly,
tamoxifen and valinomycin together were additive, implying that
tamoxifen is not a pure chloride ionophore but mediates electroneutral
chloride influx. B, effect of chloride on tamoxifen-mediated
proton permeability. Pyranine-loaded liposomes made with internal
solution of 300 mM potassium glutamate, pH 8.1, was diluted
into the same external solution in the presence or absence of tamoxifen
and 50 mM KCl as denoted in legend. At 50 s,
pHout was shifted to pH 6.9 and pHin was
followed. In the absence of chloride, tamoxifen had no effect on the
rate of pH equilibration. The presence of chloride reconstituted the
effect of tamoxifen seen in Fig. 8. C, liposomes made as in
B were diluted into 300 mM potassium glutamate
solution. Addition of 4 µM tamoxifen caused an alkaline
shift similar to Fig. 7A. But the rate of re-equilibration
was much slower than in Fig. 7A. Addition of 2 aliquots of
50 mM KCl caused increasing rate of acidification,
presumably due to Cl /H+ co-influx.
Nig, nigericin.
independently). If tamoxifen
mediates coupled transport, then chloride would be necessary for
tamoxifen-mediated proton permeability. Thus, we assessed the effect of
tamoxifen on pH in the absence of chloride. Pyranine-loaded liposomes
with a lumenal pH of 8.1 were resuspended in 300 mM
potassium glutamate and then the external pH was shifted to 6.9 (Fig.
9B). Tamoxifen had no effect on the dissipation of pH.
Inclusion of 90 mM KCl resulted in more rapid pH
equilibration (Fig. 9B). In the absence of tamoxifen,
addition of 90 mM KCl had no effect (data not shown). This
indicates that external chloride is required for tamoxifen-mediated
acidification of the lumen of the liposomes.
/H+ co-transporter, we analyzed the effect
of chloride gradients on lumenal pH in the presence of tamoxifen. In
liposomes with no lumenal chloride, addition of KCl to the external
solution results in a large chloride gradient. If tamoxifen were acting
as a coupled Cl/H+ co-transporter, the
chloride influx should mediate proton influx and lumenal acidification.
Liposomes were equilibrated to pH 8.0 both inside and out (Fig.
9C). Upon addition of tamoxifen there was a rapid step
alkaline shift of the lumen of the liposomes due to a weak base effect.
Moreover, upon each successive addition of 50 mM KCl there
was an acidic shift of the lumenal pH. This suggests that
tamoxifen-coupled HCl transport mediates the rapid re-equilibration and
explains why re-equilibration is faster when tamoxifen was added
compared with NH4Cl.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 10.
Model for tamoxifen-mediated proton
permeability. Tamoxifen is concentrated within the lipid bilayer
and exists as a charged protonated form (TNH+)
or uncharged form (TN). The uncharged form is readily
membrane-permeable, and the charged form is impermeable. This accounts
for the weak base activity of tamoxifen. In addition, TNH+
can permeate the membrane when carrying a chloride ion, accounting for
the chloride-dependent electroneutral proton
permeability.
However, a weak base effect is not sufficient to account for many of the effects of tamoxifen on transmembrane pH. Tamoxifen, but not ammonium, can increase pH equilibration rate when the lumen of the liposome is alkaline relative to the bath. Furthermore, chloride is necessary for tamoxifen-mediated proton permeability, and a chloride gradient can generate a pH gradient in the presence of tamoxifen. One possible mechanism for this process is that the permeability of the protonated form of tamoxifen increases when it is conjugated to chloride (Fig. 10).
The fact that tamoxifen inhibits acidification in so many model systems indicates that tamoxifen should affect organellar pH in many different cell types. This is consistent with the observations that tamoxifen administration has numerous physiological sequelae that are not restricted to cells expressing the estrogen receptor. Of particular significance is the observation that blocking organelle acidification through other means is sufficient to reproduce many of the effects of tamoxifen. Tamoxifen blocks bone resorption which is also blocked by antagonists of the vATPase (19). In drug-resistant tumor cells tamoxifen redistributes chemotherapeutics from the organelles to the cytoplasm (18) and increases the sensitivity of the cells to chemotherapeutics (41). These effects can be reproduced solely by mimicking the effects of tamoxifen on organellar acidification either with the use of protonophores, weak bases, or inhibitors of the vATPase (9, 22, 41). Tamoxifen decreases the rate of vesicle sorting and secretion (18) which is also seen when organelle acidification is blocked with protonophores (42). Many secreted proteins are activated by a pH-dependent proteolytic step in the Golgi. Thus, the reduced activity of many secreted proteins observed with tamoxifen treatment may also be the consequence of a tamoxifen block of organelle acidification.
Consistent with previous reports (43), we observe that tamoxifen accumulates in the lipid phase (1000:1) over the aqueous environment. Furthermore, our results suggest that membrane-bound tamoxifen is in equilibrium between a neutral and protonated form. Thus, tamoxifen would be expected to significantly perturb many properties of cellular membranes, including increased surface charge, and altered membrane tension. These effects have been reported for lipophilic weak base anesthetics (44-46). The altered membrane properties could shift the voltage dependence of many ion channels. Indeed, tamoxifen has been reported to shift the activity of many ion channels (11-16). Detailed studies on the model channel, gramicidin, have shown that membrane tension (47) and surface charge (48) are critical determinants of channel activity.
These results demonstrate that many of the effects of tamoxifen on
cells can be attributed to either membrane-active effects on organelle
acidification or surface charge. Each of these effects are independent
of the estrogen receptor. This suggests that it should be possible to
screen for other estrogen-receptor antagonists that do not also affect
organellar acidification and therefore may not share the same
physiological effects as tamoxifen.
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ACKNOWLEDGEMENTS |
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We thank Nihal Altan, Jamie M. Hahn, Judith A. Hirsch, Elliott M. Kanner, and Denise K. Marciano for valuable comments and discussion.
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FOOTNOTES |
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* This work was supported in part by American Chemical Society Grant RPG-98-177-01-CDD and National Institutes of Health Grant R01CA81257 (to S. M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Grant MSTP GM07739 at the Cornell/Rockefeller/Sloan Kettering Tri-institutional M.D./Ph.D. program.
Supported by the Pardee Foundation (Midland, MI).
** Supported by the Wolfensohn Foundation. To whom correspondence should be addressed: Laboratory of Cellular Biophysics, Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8130; Fax: 212-327-8022; E-mail: simon{at}rockvax.rockefeller.edu.
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ABBREVIATIONS |
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The abbreviations used are: vATPase, vacuolar ATPase; AO, acridine orange; DPX, p-xylene-bispyridinium bromide; POPC, palmitoyloleoyl phosphatidylcholine; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; FCCP, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; InV, inverted vesicles.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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