Goldenring, J R

Lapierre, L A

Manning, E H

Mills, J C

English

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Washington University School of Medicine Digital Commons@Becker Open Access Publications 1-1-2020 Tamoxifen acts as a parietal cell protonophore E H Manning L A Lapierre J C Mills J R Goldenring Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs RESEARCH LETTERTamoxifen Acts as aParietal CellProtonophoreOxyntic atrophy, the loss of gastricparietal cells, is a critical precursorof metaplasia in the stomach. In thecase of humans, Helicobacter pyloriinfection induces loss of parietalcells from the corpus of the stomachover years, while 6–12 months ofHelicobacter infection is required toinduce oxyntic atrophy in rodents.1In mice, acute models of parietalcell loss have helped distinguish keysteps in the induction of chief cellreprogramming into metaplasia.These studies initially used the drugDMP-777, which was demonstratedto function as a parietal cell secre-tory membrane protonophore usingassay of H/K-ATPase–dependentproton gradients in isolated rabbittubulovesicles.2 DMP-777 did notdirectly inhibit the proton pump ac-tivity. Treatment with DMP-777caused rapid parietal cell loss inboth mice and rats over 3 days,leading to development of spasmo-lytic polypeptide-expressing meta-plasia.2,3 Pretreatment of rats withproton pump inhibitors blunted theability of DMP-777 to induce oxynticatrophy.4 Similar findings were seenfor a number of molecular cousins ofDMP-777, including L635.5 WhileDMP-777 induces oxyntic atrophywithout a prominent immuneresponse, because of its coordinateaction as a neutrophil elastase in-hibitor, L635 lacks this actionagainst elastase and elicits parietalcell loss and an exuberant immuneresponse.5In more recent years, otherstudies have demonstrated the abilityof tamoxifen administration (espe-cially intraperitoneal doses of 5 mgor greater) to also induce parietal cellloss.6 As with DMP-777, pretreat-ment of mice with proton pump in-hibitors ameliorated the effects oftamoxifen to induce parietal cellloss.6 However, no studies havedirectly assayed the effects oftamoxifen as a parietal cell proto-nophore. We have therefore sought tocompare the effects of tamoxifen withthose of DMP-777 and L635 on acidsequestration in parietal celltubulovesicles.Parietal cell tubulovesicles wereisolated from rabbit stomach mucosausing standard protocols and tubulove-sicles layering above 20% sucrose ingradient centrifugation were utilized asthe tightest tubulovesicle membranes(see supplementary methods).7 Thefluorescence of acridine orange wasassayed in tubulovesicles using induc-tion of pumping in the presence ofadenosine triphosphate and valinomy-cin. Activation of acid pumping intotubulovesicles causes a rapid quenchingof acridine orange fluorescence indica-tive of pumping of acid into the lumenof tubulovesicles (Figure 1). After theestablishment of the proton gradient intubulovesicles, tamoxifen, DMP-777, orL635 were added in concentrationsfrom 0.1 to 10 mM and effects on thequenching of acridine orange wereassayed (Figure 1). At concentrationsfrom 1 to 10 mM, all 3 drugs caused arapid decrease in acridine orange fluo-rescence, indicative of disruption of theproton gradient. These findings sug-gested that all 3 drugs act similarly asprotonophores in parietal cell acidsecretory membranes.The results in these studiesconfirm that tamoxifen does act as aparietal cell protonophore, which canlead to back wash of acid intosecreting parietal cells, the likelycause of parietal cell death followingadministration of high-dose tamox-ifen. The potency of tamoxifen as aparietal cell protonophore is similarto that both DMP-777 and L635. Itshould be noted that a number ofhydrophobic drugs with properties ofweak bases may have parietal cellprotonophore activities. KN-93, whichhas been utilized as an inhibitor ofcalmodulin-dependent protein kinaseII, has a strong protonophore effecton parietal cells.8 Nevertheless, itshould be noted that theseprotonophore actions requireextremely high doses of drug that areusually greater than an order ofmagnitude higher than doses used fortreating patients.6Nevertheless, because tamoxifen-inducible Cre recombinase strainsare commonly used, especially totrack stem cell activity, it is critical torecognize that tamoxifen can kill pa-rietal cells, triggering adaptive, non-homeostatic patterns of proliferationin the stomach. A recent report bySamuelson et al9 has highlighted thedose dependence of tamoxifen actionin the stomach. That report noted agap in the knowledge of the precisemechanism of tamoxifen actionrelated to oxyntic atrophy leading usto perform the present studies. It isnotable that the effects of tamoxifento induce parietal cell loss, as well asthe recovery from tamoxifen, werenot dependent on gastrin. Similar re-sults were previously observed withDMP-777 administration.3 Thus,cholinergic stimulation in these ani-mals may be adequate to maintainsufficient acid secretion and lead toacute parietal cell loss. While tamox-ifen, DMP-777, and L635 all possess aparietal cell protonophore capacity, itremains unclear as to whether theiroff-target effects may differ. It is clearthat these 3 models of acute oxynticatrophy all differ in the range of in-flammatory reactions they incite.Given the recent evidence for theroles of immune cells in promotingthe induction and progression ofmetaplasia,10 each of these modelsneeds to be evaluated in detail, and incomparison with more chronic formsof oxyntic atrophy.E. H. MANNING1,2,3L. A. LAPIERRE1,2,3J. C. MILLS4,5,6,7J. R. GOLDENRING1,2,3,81Department of Surgery, Vanderbilt University Med-ical Center, Nashville, Tennessee2Epithelial Biology Center, Vanderbilt UniversityMedical Center, Nashville, Tennessee3Nashville VA Medical Center, Nashville, Tennessee4Division of Gastroenterology, Washington Univer-sity School of Medicine, Washington University in St.Louis, St. Louis, MissouriDownloaded for Anonymous User (n/a) at Washington University in Saint Louis Bernard Becker Medical Library from ClinicalKey.com by Elsevier on October 08, 2020.For personal use only. No other uses without permission. Copyright ©2020. Elsevier Inc. All rights reserved.Figure 1. Assay of acridine orangefluorescence in isolated tubulove-sicles treated with tamoxifen, DMP-777, or L635. Isolated rabbit gastrictubulovesicles were incubated with acri-dine orange, and initiation of the protonpumping gradient was activated withaddition of valinomycin and adenosinetriphosphate (first black arrowhead).Control (no addition), vehicle solutions,or drugs were then added at the indi-cated concentrations after 15 minutes(second black arrowhead) anddequenching was analyzed for 20 min.The traces (± SD) represent representa-tive experiments from 4 separateanalyses. (A) Tamoxifen response. (B)DMP-777 response. (C) L635 response.(D) Comparison of tamoxifen, DMP-777,and L635 response at 10 mM. Note thatdimethyl sulfoxide (DMSO) vehicle con-trol samples showed a small baselineadjustment likely due to the time requiredfor addition of drugs.656 Manning et al Cellular and Molecular Gastroenterology and Hepatology Vol. 10, No. 3Downloaded for Anonymous User (n/a) at Washington University in Saint Louis Bernard Becker Medical Library from ClinicalKey.com by Elsevier on October 08, 2020.For personal use only. No other uses without permission. Copyright ©2020. Elsevier Inc. All rights reserved.5Department of Internal Medicine, Washington Uni-versity School of Medicine, Washington University inSt. Louis, St. Louis, Missouri6Department of Developmental Biology, WashingtonUniversity School of Medicine, Washington Univer-sity in St. Louis, St. Louis, Missouri7Department of Pathology and Immunology, Wash-ington University School of Medicine, WashingtonUniversity in St. Louis, St. Louis, Missouri8Department of Cell and Developmental Biology,Vanderbilt University Medical Center, Nashville,TennesseeAddress correspondence to: James R. Goldenring,MD, PhD, Epithelial Biology Center, VanderbiltUniversity Medical Center, 10435 Medical ResearchBuilding IV, 2213 Garland Avenue, Nashville, TN37232. e-mail: jim.goldenring@vumc.org; fax:(615) 343-1591.References1. Fox JG, et al. Gastroenterology 1996;110:155–166.2. Goldenring JR, et al. Gastroenterology 2000;118:1080–1093.3. Nomura S, et al. Am J Physiol GastrointestLiver Physiol 2004;288:G362–G375.4. Ogawa M, et al. Dig Dis Sci 2006;51:431–439.5. Nam KT, et al. Gastroenterology 2010;139:2028–2037.6. Huh WJ, et al. Gastroenterology 2012;142:21–24.e7.7. Crothers JM Jr, et al. Am J Physiol 1993;265:G231–G241.8. Mamiya N, et al. Biochem Biophys ResCommun 1993;195:608–615.9. Keeley TM, et al. Cell Mol GastroenterolHepatol 2019;8:365–367.10. Petersen CP, et al. Gastroenterology 2014;146:1727–1738.Most current article© 2020 The Authors. Published by ElsevierInc. on behalf of the AGA Institute. This isan open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).2352-345Xhttps://doi.org/10.1016/j.jcmgh.2020.04.012Received December 10, 2019. Accepted April24, 2020.CRediT Authorship ContributionsElizabeth Manning (Data curation: Lead; Formalanalysis: Equal; Methodology: Lead; Writing –review & editing: Supporting) Lapierre A. Lapierre(Data curation: Equal; Formal analysis: Equal;Methodology: Equal; Writing – review & editing:Supporting) Jason C. Mills (Conceptualization:Equal; Writing – review & editing: Supporting)James R. Goldenring, MD, PhD(Conceptualization: Lead; Methodology: Equal;Project administration: Lead; Supervision: Lead;Writing – original draft: Lead)Conflicts of interestThe authors disclose no conflictsFundingThese studies were supported by Department ofVeterans Affairs Merit Review Award Grant No.IBX000930 (to James R. Goldenring); NationalInstitutes of Health Grant Nos. RO1 DK071590 (toJames R. Goldenring), RO1 DK101332 (to JamesR. Goldenring), R01 DK094989 (to Jason C. Mills),RO1 DK105129 (to Jason C. Mills), and RO1DK110406 (to Jason C. Mills); and core resourcesof the Vanderbilt Digestive Disease Center(National Institutes of Health P30 DK058404).2020 Parietal Cell Protonophore 657Downloaded for Anonymous User (n/a) at Washington University in Saint Louis Bernard Becker Medical Library from ClinicalKey.com by Elsevier on October 08, 2020.For personal use only. No other uses without permission. Copyright ©2020. Elsevier Inc. All rights reserved.Supplementary Table 1.Formulation of Test CompoundsControl or sample name(reagent added at timepoint 3)Control or sampledetailsReagentstorage stockReagent solution preparedfor assayFinal reagentconcentrationin assay wellDiluent only Negative control forassayAcridine orange diluentDMSO Negative control forDMP-777, L635,and tamoxifen2.5% DMSO in acridineorange diluent0.25%DMP-777 Positive control 10 mM in DMSO 100 mM in acridine orangediluent10, 3, or 1 mML635 Positive control 11 mM in DMSO 100 mM in acridine orangediluent10, 3, or 1 mMTamoxifen Sample 4 mM in DMSO 100 mM in acridine orangediluent10, 3, 1, 0.3, or0.1 mMDMSO, dimethyl sulfoxide.Supplementary Materialsand MethodsMaterialsDMP-777 was a gift of DuPont-MerckCorporation (North Billerica, MA).L635 was synthesized in the Vander-bilt Chemical Biology Shared Resourceand was validated as a single enan-tiomeric compound of >99.5% purity.Tamoxifen was obtained from Sigma-Aldrich (St. Louis, MO). All othercompounds were obtained fromSigma-Aldrich.Preparation of Rabbit GastricTubulovesiclesRabbit tubulovesicles were preparedfrom homogenized gastric mucosa bydifferential centrifugation followedby sucrose gradient velocity sedi-mentation, as previously described.1Tubulovesicle membranes that floa-ted on the 20% sucrose cushionwere utilized as high-resistancetubulovesicles.Assay of Acridine OrangeAccumulation IntoTubulovesiclesAssay master mix was composed of 1.2-mM acridine orange/290-mM adenosinetriphosphate/24 mg/mL tubulovesiclesfroma freshly thawedaliquot in acridineorange diluent (150-mM sucrose/75-mM KCl/250-mM MgSO4/5-mM pipera-zine-N,N0-bis(2-ethanesulfonic acid)/5-mM Tris, pH 7). Valinomycin wasdiluted to 20 mM in acridine orangediluent from a 0.9-mM stock in dimethylsulfoxide. Control and sample assay re-agents were diluted as described inSupplementary Table 1.Master mix (85 mL) was added towells in a 96-well microtiter plate inquadruplicate for each control or sam-ple. The plate was read (time point 1)using a BioTek Synergy 4 (BioTek,Winooski, VT) plate reader at 493-nmexcitation/530-nm emission once perminute for 3 minutes at ambient tem-perature, then ejected. Valinomycin (5mLof 20-mMsolution)was added to eachwell to a final concentration of 1 mM(time point 2), then the platewas shakenfor 5 seconds and read for 10 more mi-nutes and ejected. Control and samplereagents diluted in acridine orangediluent (10 mL each) were added to thewells to the final concentrationsdescribed in Supplementary Table 1(time point 3). The total volume in eachwell after time point 3 was 100 mL. Theplate was shaken for 5 seconds and readfor at least 15 more minutes.Data AnalysisRaw absorbance data for each wellwere converted to fold change bydividing all absorbance readings by thefirst absorbance reading using GoogleSheets. Fold-change means and stan-dard deviations for each control andsample across the quadruplicates werealso calculated. Figures were madeusing Open Office Calc (Apache Soft-ware Foundation, Forest Hill, MD).Reference1. Lapierre LA, et al. Am J Physiol GastrointestLiver Physiol 2007;292:G1249–G1262.657.e1 Manning et al Cellular and Molecular Gastroenterology and Hepatology Vol. 10, No. 3Downloaded for Anonymous User (n/a) at Washington University in Saint Louis Bernard Becker Medical Library from ClinicalKey.com by Elsevier on October 08, 2020.For personal use only. No other uses without permission. Copyright ©2020. Elsevier Inc. All rights reserved.

Tamoxifen acts as a parietal cell protonophore

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Tamoxifen acts as a parietal cell protonophore

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