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• W2134595594 abstract "Tamoxifen has been reported to inhibit acidification of cytoplasmic organelles in mammalian cells. Here, the mechanism of this inhibition is investigated using in vitroassays 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 (V m) 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 has been reported to inhibit acidification of cytoplasmic organelles in mammalian cells. Here, the mechanism of this inhibition is investigated using in vitroassays 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 (V m) 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 (1Jaiyesimi I.A. Buzdar A.U. Decker D.A. Hortobagyi G.N. J. Clin. Oncol. 1995; 13: 513-529Crossref PubMed Google Scholar). In addition, it is currently being considered for widespread use in healthy women for breast cancer prevention (2Jordan V.C. Proc. Soc. Exp. Biol. Med. 1995; 208: 144-149Crossref PubMed Scopus (51) Google Scholar, 3Fisher B. Costantino J.P. Wickerham D.L. Redmond C.K. Kavanah M. Cronin W.M. Vogel V. Robidoux A. Dimitrov N. Atkins J. Daly M. Wieand S. Tan-Chiu E. Ford L. Wolmark N. J. Natl. Cancer Inst. 1998; 90: 1371-1388Crossref PubMed Scopus (4872) Google Scholar). 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 (5Chatterjee M. Harris A.L. Br. J. Cancer. 1990; 62: 712-717Crossref PubMed Scopus (39) Google Scholar, 6Berman E. Adams M. Duigou-Osterndorf R. Godfrey L. Clarkson B.A.M. Blood. 1991; 77: 818-825Crossref PubMed Google Scholar, 7Berman E. McBride M. Lin S. Menedez-Botet C. Tong W. Leukemia (Balt.). 1995; 9: 1631-1637PubMed Google Scholar, 8Weinlander G. Kornek G. Raderer M. Hejna M. Tetzner C. Scheithauer W. J. Cancer Res. Clin. Oncol. 1997; 123: 452-455Crossref PubMed Google Scholar, 9Altan N. Chen Y. Schindler M. Simon S.M. J. Exp. Med. 1998; 187: 1583-1598Crossref PubMed Scopus (248) Google Scholar), inhibit bone resorption and osteoporosis both in vivo and in vitro (10Love R.R. Mazess R.B. Barden H.S. Epstein S. Newcomb P.A. Jordan V.C. Carbone P.P. DeMets D.L. N. Engl. J. Med. 1992; 326: 852-856Crossref PubMed Scopus (1013) Google Scholar), and inhibit a number of channels, including the volume activated chloride channel (11Zhang J.J. Jacob T.J. Valverde M.A. Hardy S.P. Mintenig G.M. Sepulveda F.V.G.D. Hyde S.C. Trezise A.E. Higgins C.F. J. Clin. Invest. 1994; 94: 1690-1697Crossref PubMed Google Scholar, 12Ehring G.R. Osipchuk Y.V. Cahalan M.D. J. Gen. Physiol. 1994; 104: 1129-1161Crossref PubMed Scopus (74) Google Scholar) and calcium channels (13Greenberg D.A. Carpenter C.L. Messing R.O. Cancer Res. 1987; 47: 70-74PubMed Google Scholar, 14Song J. Standley P.R. Zhang F. Joshi D. Gappy S. Sowers J.R. Ram J.L. J. Pharmacol. Exp. Ther. 1996; 277: 1444-1453PubMed Google Scholar, 15Williams J.P. Blair H.C. McKenna M.A. Jordan S.E. McDonald J.M. J. Biol. Chem. 1996; 271: 12488-12495Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 16Turner R.T. Wakley G.K. Hannon K.S. Bell N.H. Endocrinology. 1988; 122: 1146-1150Crossref PubMed Scopus (204) Google Scholar). These effects have been attributed to inhibition of P-glycoprotein (17Callaghan R. Higgins C.F. Br. J. Cancer. 1995; 71: 294-299Crossref PubMed Scopus (103) Google Scholar), calmodulin (15Williams J.P. Blair H.C. McKenna M.A. Jordan S.E. McDonald J.M. J. Biol. Chem. 1996; 271: 12488-12495Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and direct channel interaction (11Zhang J.J. Jacob T.J. Valverde M.A. Hardy S.P. Mintenig G.M. Sepulveda F.V.G.D. Hyde S.C. Trezise A.E. Higgins C.F. J. Clin. Invest. 1994; 94: 1690-1697Crossref PubMed Google Scholar), respectively. Previously, we have observed that tamoxifen inhibits acidification of intracellular organelles of both estrogen receptor positive and negative cell lines (18Altan N. Chen Y. Schindler M. Simon S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4432-4437Crossref PubMed Scopus (100) Google Scholar). This inhibition of acidification may be a mechanism for many of the effects of tamoxifen. For example, the effects of tamoxifen on osteoporosis (19Sundquist K. Lakkakorpi P. Wallmark B. Vaananen K. Biochem. Biophys. Res. Commun. 1990; 168: 309-313Crossref PubMed Scopus (154) Google Scholar), vesicular transport (20Gekle M. Mildenberger S. Freudinger R. Silbernagl S. Am. J. Physiol. 1995; 268: F899-F906PubMed Google Scholar,21van Weert A.W. Dunn K.W. Gueze H.J. Maxfield F.R. Stoorvogel W. J. Cell Biol. 1995; 130: 821-834Crossref PubMed Scopus (302) Google Scholar), or multidrug resistance (9Altan N. Chen Y. Schindler M. Simon S.M. J. Exp. Med. 1998; 187: 1583-1598Crossref PubMed Scopus (248) Google Scholar, 22Hurwitz S.J. Terashima M. Mizunuma N. Slapak C.A. Blood. 1997; 89: 3745-3754Crossref PubMed Google Scholar) are mimicked by blocking the proton vATPase 1The 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 cyanidep-trifluoromethoxy-phenylhydrazone; InV, inverted vesicles. 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 V m 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 (18Altan N. Chen Y. Schindler M. Simon S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4432-4437Crossref PubMed Scopus (100) Google Scholar). 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. 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). 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 mmEDTA, and 1× protease inhibitor mix (1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 16 μmphenylmethylsulfonyl 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 mmHEPES, 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 minterface. To monitor acidification of the total microsomal fraction, the quenching of AO fluorescence was monitored essentially as described previously (23Barasch J. Kiss B. Prince A. Saiman L. Gruenert D. Al-Awqati Q. Nature. 1991; 352: 70-73Crossref PubMed Scopus (426) Google Scholar). 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 λex = 488 nm and λem = 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 mmNaN3), with 6 μm AO (5 mm stock in H2O) in a cuvette. To examine the ability of vesicles to generate a ΔpH 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 mmTris-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 ΔpH formation. To study the effects of tamoxifen and bafilomycin A1 on vesicles with a pre-existing ΔpH tamoxifen or bafilomycin A1 were added 10 min after the addition of Tris-ATP. 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 λem = 520 nm as described previously (18Altan N. Chen Y. Schindler M. Simon S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4432-4437Crossref PubMed Scopus (100) Google Scholar). Vacuoles from S.cerevisiae were prepared from the protease-deficient strainBJ2407 (Yeast Genetic Stock Center, University of California, Berkeley) by sequential flotation through 12 and 8% Ficoll 400 cushion as described previously (24Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods Enzymol. 1991; 194: 644-661Crossref PubMed Scopus (287) Google Scholar) 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 α-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. InV were prepared from the DH5α strain as described (25Simon S.M. Blobel G. Cell. 1992; 69: 677-684Abstract Full Text PDF PubMed Scopus (170) Google Scholar). Acidification was measured using AO as described above. Oxonol V is a membrane-permeable anionic fluorescent probe that accumulates into the inner leaflet of vesicles with positive V m, resulting in quenching of fluorescence. Vacuoles were suspended in chloride or gluconate vesicle buffer with 1 μm oxonol V. Fluorescence was measured with a λex = 600 nm and λem = 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 V m was manifested in fluorescence quenching. 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 A 660 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. The lumenal pH (pHL) of liposomes was assayed with pyranine, a fluorescent dye with a pK a ∼7.3 and a λex = 405 nm in its acid form (−3 charge) and a λex = 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. 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 λem = 510 nm was monitored with dual excitation wavelengths of λex = 405 and λex = 455 nm. Sequential aliquots of 0.1 mmglycylglycine, 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 χ2 >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. 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 mmglycylglycine, 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(λex = 405 nm/λex = 405 nm) + c, where x andc 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. 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. Lucigenin is a fluorescent dye that is collisionally quenched by chloride and other halides but not by nitrate (26Biwersi J. Tulk B. Verkman A.S. Anal. Biochem. 1994; 219: 139-143Crossref PubMed Scopus (114) Google Scholar). 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 mmKNO3, 10 mm K-HEPES, pH 7.3) with 1 μm tributyltin (TBT) a Cl−-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 (λex = 370 nm/λex = 505 nm) was recorded. The fluorescence was fitted to the Stern-Volmer equation:F 0/F = 1 + k[Cl], where F 0 is the fluorescence in the absence of chloride. 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 kcalculated from the titration curve. The concentration of tamoxifen was measured by its absorbance peak at 245 nm. TheA 245 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 A 245 of the aqueous phase was acquired. 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 (23Barasch J. Kiss B. Prince A. Saiman L. Gruenert D. Al-Awqati Q. Nature. 1991; 352: 70-73Crossref PubMed Scopus (426) Google Scholar). 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. 1 A, att = 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.1 A). Inhibition was evident when using 1 μmtamoxifen, 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. 1 A, inset). The ID50 for maximal quenching is approximately 3 μm, which is in the same range that tamoxifen inhibits acidification in vivo (18Altan N. Chen Y. Schindler M. Simon S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4432-4437Crossref PubMed Scopus (100) Google Scholar). As a positive control, we employed bafilomycin A1, a potent and specific inhibitor of the vATPase responsible for acidification of all intracellular compartments (27Bowman E.J. Siebers A. Altendorf K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7972-7976Crossref PubMed Scopus (1605) Google Scholar). To determine the time course for the inhibition of acidification by tamoxifen, the drug was added 10 min after addition of ATP (Fig.1 B). 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.1 A). Addition of nigericin (Fig. 1 B) 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. 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.1 C). 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. Acidification of intracellular organelles utilizes an electrogenic proton pump (the vATPase) and chloride channels (28Glickman J. Croen K. Kelly S. Al-Awqati Q. J. Cell Biol. 1983; 97: 1303-1308Crossref PubMed Scopus (188) Google Scholar, 29Mellman I. Fuchs R. Helenius A. Annu. Rev. Biochem. 1986; 55: 663-700Crossref PubMed Google Scholar). The vATPase couples ATP hydrolysis to proton movement. The unidirectional movement of the proton generates an inside positive V m which limits acidification. The chloride channels allow passive chloride influx into the organelles, dissipating the V m. Tamoxifen could inhibit acidification by the following possible mechanisms: direct inhibition of the vATPase; indirect inhibition of the vATPase through modulation of the V m (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. 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 (30Williams J.P. McDonald J.M. McKenna M.A. Jordan S.E. Radding W. Blair H.C. J. Cell. Biochem. 1997; 66: 358-369Crossref PubMed Scopus (18) Google Scholar). Tamoxifen has been reported to inhibit the volume-activated chloride channel (11Zhang J.J. Jacob T.J. Valverde M.A. Hardy S.P. Mintenig G.M. Sepulveda F.V.G.D. Hyde S.C. Trezise A.E. Higgins C.F. J. Clin. Invest. 1994; 94: 1690-1697Crossref PubMed Google Scholar). 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 pK a of 6.9 when measured by NMR in 10% Triton solution (31Bottega R. Epand R.M. Biochemistry. 1992; 31: 9025-9030Crossref PubMed Scopus (130) Google Scholar). 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. Tamoxifen partitions into lipids, increases membrane fluidity, and decreases lipid peroxidation (32Wiseman H. Quinn P. Halliwell B. FEBS Lett. 1993; 330: 53-56Crossref PubMed Scopus (109) Google Scholar). 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 (33Garlid K.D. Nakashima R.A. J. Biol. Chem. 1983; 258: 7974-7980Abstract Full Text PDF PubMed Google Scholar, 34Sun X. Garlid K.D. J. Biol. Chem. 1992; 267: 19147-19154Abstract Full Text PDF PubMed Google Scholar). Each of these potential mechanisms has distinct consequences for ATPase activity and V m of the acidic organelle (TableI). If tamoxifen inhibits the vATPase, it would decrease the ATPase activity. In addition, it should decreaseV m of the organelles since the proton pumping is generating the V m. If tamoxifen inhibits the chloride channel, it would increase V m, since the chloride channel serves to dissipate V m. As a consequence of the increased V m, the vATPase cannot pump protons, resulting in a decreased rate of ATP hydrolysis. If tamoxifen is a protonophore, it should decrease V mby 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 V m and ATPase activity since it dissipates the proton gradient in favor of an electrical gradient.Table IPredicted effects of potential mechanisms of tamoxifen on Vm and ATPase activityV mATPase activityInhibit H-ATPaseDecreaseDecreaseBlock counter-ion transportIncreaseDecreaseIncrease proton permeabilityDecreaseIncreaseWeak base effectSame to slight increaseSlight increase Open table in a new tab The predictions of these mechanisms were tested on isolated vacuoles from S. cerevisiae. Vacuoles from S. cerevisiaeoff" @default.
• W2134595594 created "2016-06-24" @default.
• W2134595594 creator A5001303396 @default.
• W2134595594 creator A5036195107 @default.
• W2134595594 creator A5052676364 @default.
• W2134595594 date "1999-06-01" @default.
• W2134595594 modified "2023-09-29" @default.
• W2134595594 title "A Mechanism for Tamoxifen-mediated Inhibition of Acidification" @default.
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