The Role of Carbonic Anhydrase 9 in Regulating Extracellular and Intracellular pH in Three-dimensional Tumor Cell Growths
2009; Elsevier BV; Volume: 284; Issue: 30 Linguagem: Inglês
10.1074/jbc.m109.006478
ISSN1083-351X
AutoresPawel Swietach, Shalini Patiar, Claudiu T. Supuran, Adrian L. Harris, Richard D. Vaughan‐Jones,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoWe have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of ∼300 μm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pHi) and pH in the restricted extracellular spaces (pHe). With no CA9 expression, spheroids developed very low pHi (∼6.3) and reduced pHe (∼6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pHi = ∼6.6), whereas extracellular acidity was enhanced (pHe = ∼6.6), so that radial pHi gradients were smaller and radial pHe gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pHe indicates the enzyme is facilitating CO2 excretion from cells (by converting vented CO2 to extracellular H+), rather than facilitating membrane H+ transport (such as H+ associated with metabolically generated lactic acid). This latter process requires titration of exported H+ ions with extracellular HCO3−, which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pHe will depend on the cellular CO2/lactic acid emission ratio (set by local oxygenation and membrane HCO3− uptake). Our results suggest that CO2-producing tumors may express CA9 to facilitate CO2 excretion, thus raising pHi and reducing pHe, which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity. We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of ∼300 μm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pHi) and pH in the restricted extracellular spaces (pHe). With no CA9 expression, spheroids developed very low pHi (∼6.3) and reduced pHe (∼6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pHi = ∼6.6), whereas extracellular acidity was enhanced (pHe = ∼6.6), so that radial pHi gradients were smaller and radial pHe gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pHe indicates the enzyme is facilitating CO2 excretion from cells (by converting vented CO2 to extracellular H+), rather than facilitating membrane H+ transport (such as H+ associated with metabolically generated lactic acid). This latter process requires titration of exported H+ ions with extracellular HCO3−, which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pHe will depend on the cellular CO2/lactic acid emission ratio (set by local oxygenation and membrane HCO3− uptake). Our results suggest that CO2-producing tumors may express CA9 to facilitate CO2 excretion, thus raising pHi and reducing pHe, which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity. The carbonic anhydrases (CAs) 3The abbreviations used are: CAcarbonic anhydraseDOG2-deoxy-d-glucoseFSfluorescein 5(6)-sulfonic acidDFN-(fluorescein 5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamineAP1051-([4-sulfamoylphenyl)ethyl]-2,4,6-trimethyl pyridinium perchlorateATZacetazolamideEVempty vectorROIregion of interestAMC7-amino-4-methylcoumarinFCCPcarbonyl cyanide p-trifluoromethoxyphenylhydrazoneMCTmonocarboxylic acid transporterMes4-morpholineethanesulfonic acidDIDS4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. are a family of enzymes that reversibly catalyze CO2 hydration to H+ and HCO3− (1Supuran C.T. Nat. Rev. Drug Discov. 2008; 7: 1-14Crossref PubMed Scopus (2540) Google Scholar, 2Swietach P. Vaughan-Jones R.D. Harris A.L. Cancer Metastasis Rev. 2007; 26: 299-310Crossref PubMed Scopus (426) Google Scholar). Recent studies have identified several CA isoforms, such as CA4, CA9, CA12, and CA14, with extracellular-facing catalytic sites (2Swietach P. Vaughan-Jones R.D. Harris A.L. Cancer Metastasis Rev. 2007; 26: 299-310Crossref PubMed Scopus (426) Google Scholar). Many cells express extracellular CA (CAe) isoforms, but their physiological role remains unclear. In particular, the strong link between cancer and CA9 expression (1Supuran C.T. Nat. Rev. Drug Discov. 2008; 7: 1-14Crossref PubMed Scopus (2540) Google Scholar, 2Swietach P. Vaughan-Jones R.D. Harris A.L. Cancer Metastasis Rev. 2007; 26: 299-310Crossref PubMed Scopus (426) Google Scholar, 3Chiche J. Ilc K. Laferrière J. Trottier E. Dayan F. Mazure N.M. Brahimi-Horn M.C. Pouysségur J. Cancer Res. 2009; 69: 358-368Crossref PubMed Scopus (584) Google Scholar, 4Pastorek J. Pastoreková S. Callebaut I. Mornon J.P. Zelník V. Opavský R. Zat'ovicová M. Liao S. Portetelle D. Stanbridge E.J. Závada J. Burny A. Kettmann R. Oncogene. 1994; 9: 2877-2888PubMed Google Scholar, 5Wykoff C.C. Beasley N.J. Watson P.H. Turner K.J. Pastorek J. Sibtain A. Wilson G.D. Turley H. Talks K.L. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Harris A.L. Cancer Res. 2000; 60: 7075-7083PubMed Google Scholar) has provoked great interest in the role of CAe in tumor biology. carbonic anhydrase 2-deoxy-d-glucose fluorescein 5(6)-sulfonic acid N-(fluorescein 5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine 1-([4-sulfamoylphenyl)ethyl]-2,4,6-trimethyl pyridinium perchlorate acetazolamide empty vector region of interest 7-amino-4-methylcoumarin carbonyl cyanide p-trifluoromethoxyphenylhydrazone monocarboxylic acid transporter 4-morpholineethanesulfonic acid 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Based on their topology, CAe isoforms are likely to regulate the concentration of extracellular H+, CO2, and HCO3−. Cell metabolism drives transmembrane fluxes of H+ ions, CO2 and HCO3−, and can provide substrate for the CAe-assisted reaction. For example, CO2 is released from aerobically respiring cells. By consuming or producing H+ ions, the CAe-catalyzed reaction will affect extracellular pH (pHe). Many membrane proteins are modulated by pHe (6Ludwig M.G. Vanek M. Guerini D. Gasser J.A. Jones C.E. Junker U. Hofstetter H. Wolf R.M. Seuwen K. Nature. 2003; 425: 93-98Crossref PubMed Scopus (527) Google Scholar, 7Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N.Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (180) Google Scholar, 8Huang W.C. Swietach P. Vaughan-Jones R.D. Ansorge O. Glitsch M.D. Curr. Biol. 2008; 18: 781-785Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Some of these are acid/base transporters that regulate intracellular pH (pHi) (9Boron W.F. Adv. Physiol. Educ. 2004; 28: 160-179Crossref PubMed Scopus (251) Google Scholar). Such modulation allows pHe to cross-talk with pHi (10Jean T. Frelin C. Vigne P. Barbry P. Lazdunski M. J. Biol. 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Extracellular pH can also affect tissue structure through the release or modulation of proteolytic enzymes that act on the extracellular matrix (14Gatenby R.A. Gawlinski E.T. Gmitro A.F. Kaylor B. Gillies R.J. Cancer Res. 2006; 66: 5216-5223Crossref PubMed Scopus (592) Google Scholar, 15Kato Y. Nakayama Y. Umeda M. Miyazaki K. J. Biol. Chem. 1992; 267: 11424-11430Abstract Full Text PDF PubMed Google Scholar). In addition, the pHe-pHi difference is important in determining the distribution of membrane-permeant weak acids/bases, which include many drugs used clinically (e.g. doxorubicin). A complete understanding of pH regulation at tissue level requires characterization of events occurring within cells, at their surface membrane, and in the surrounding extracellular space. To date, many pH studies have treated the extracellular space as an infinite, well-stirred, and equilibrated compartment of constant pH. This condition is compatible with experimentally superfused, isolated cells, but it may not apply to all cells in situ. Blood plasma is a major component of extracellular fluid. In health, plasma pH is regulated to ∼7.4 by the lungs and kidneys, acting in concert to remove excess acid/base that has been added to blood from dietary or cellular sources. Tissue fluid occupies the gap between plasma and cells (with the exception of blood-borne cells). Under conditions of ideal diffusive coupling between cells and capillaries, pHe in tissue fluid would be held close to plasma pH. However, pHe close to the cell surface can diverge from 7.4, particularly when the cell-capillary distance is increased (e.g. as a result of poor blood perfusion), when the excreted acid/base load is elevated, or when the local buffering capacity is compromised. Regulation of pHe is particularly important in tumors because these are characterized by a high metabolic rate (16Gatenby R.A. Gillies R.J. Nat. Rev. Cancer. 2004; 4: 891-899Crossref PubMed Scopus (3723) Google Scholar, 17Gillies R.J. Robey I. Gatenby R.A. J. Nucl. Med. 2008; 49: 24S-42SCrossref PubMed Scopus (483) Google Scholar) and abnormal blood perfusion (18Vaupel P. Kallinowski F. Okunieff P. Cancer Res. 1989; 49: 6449-6465PubMed Google Scholar, 19Stubbs M. McSheehy P.M. Griffiths J.R. Bashford C.L. Mol. Med. Today. 2000; 6: 15-19Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar). Studies have shown that tumors develop low pHe (∼6.9) in response to the mismatch between metabolic demand and the capacity to remove metabolic waste products (14Gatenby R.A. Gawlinski E.T. Gmitro A.F. Kaylor B. Gillies R.J. Cancer Res. 2006; 66: 5216-5223Crossref PubMed Scopus (592) Google Scholar, 18Vaupel P. Kallinowski F. Okunieff P. Cancer Res. 1989; 49: 6449-6465PubMed Google Scholar, 20Gillies R.J. Raghunand N. Karczmar G.S. Bhujwalla Z.M. J. Magn. Reson. Imaging. 2002; 16: 430-450Crossref PubMed Scopus (459) Google Scholar). Tumors can survive in considerably more acidic interstitium than their non-neoplastic counterparts, partly because of their ability to maintain a favorably alkaline pHi for growth and development (21Griffiths J.R. Stevens A.N. Iles R.A. Gordon R.E. Shaw D. Biosci. Rep. 1981; 1: 319-325Crossref PubMed Scopus (116) Google Scholar). It has been argued that tumors can survive selectively by maintaining a level of pHe that is lethal to normal cells but not sufficiently acidic to kill the tumor itself (2Swietach P. Vaughan-Jones R.D. Harris A.L. Cancer Metastasis Rev. 2007; 26: 299-310Crossref PubMed Scopus (426) Google Scholar, 14Gatenby R.A. Gawlinski E.T. Gmitro A.F. Kaylor B. Gillies R.J. Cancer Res. 2006; 66: 5216-5223Crossref PubMed Scopus (592) Google Scholar, 22Griffiths J.R. McIntyre D.J. Howe F.A. Stubbs M. Novartis Found. Symp. 2001; 240: 46-62Crossref PubMed Google Scholar). A major fraction of cell-derived acid is excreted in the form of CO2, generated directly from the Krebs cycle or from titration of intracellular H+ with HCO3−. To maintain a steep outward gradient for CO2 excretion, extracellular CO2 must not accumulate. This can be achieved by venting CO2 to the nearest capillary or by reacting CO2 locally to produce H+ and HCO3−. The balance between these two fluxes is set by the diffusion distance and CO2 hydration kinetics, respectively. Diffusion is anecdotally considered to be fast. However, over long distances, CO2 diffusion may be slower than its local reactive flux. Assuming a CO2 diffusion coefficient, DCO2, of 2500 μm2/s and a spontaneous CO2 hydration rate, kf, of 0.14 s−1 (23Swietach P. Wigfield S. Cobden P. Supuran C.T. Harris A.L. Vaughan-Jones R.D. J. Biol. Chem. 2008; 283: 20473-20483Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), local CO2 consumption by reaction will be faster than CO2 diffusion over distances >190 μm (√(2 × DCO2/kf)). The reactive flux can be augmented enzymatically by CAe, to increase further the importance of reactive versus diffusive consumption of CO2. If, for instance, hydration is catalyzed 10-fold, reactive CO2 removal would exceed diffusive CO2 removal over distances of >60 μm. The remainder of transmembrane acid efflux takes the form of lactic acid, generated from anaerobic respiration or aerobic glycolysis (Warburg effect) (16Gatenby R.A. Gillies R.J. Nat. Rev. Cancer. 2004; 4: 891-899Crossref PubMed Scopus (3723) Google Scholar). Lactic acid efflux can be accelerated if its extracellular concentration is kept low by diffusive dissipation or by CAe-catalyzed extracellular titration of H+ with HCO3−. It is important to note that CAe-catalyzed hydration of extracellular CO2 will reduce pHe, whereas titration of extracellular lactic acid by HCO3− (to form CO2, a weaker acid) will raise pHe. Therefore, the capacity of CAe to regulate pHe will depend on the chemistry of the excreted acid. In most healthy tissues at rest, the majority of cellular acid is emitted as CO2. Recent work on tumors also suggests a dominance of CO2 over lactic acid (22Griffiths J.R. McIntyre D.J. Howe F.A. Stubbs M. Novartis Found. Symp. 2001; 240: 46-62Crossref PubMed Google Scholar, 24Holm E. Hagmüller E. Staedt U. Schlickeiser G. Günther H.J. Leweling H. Tokus M. Kollmar H.B. Cancer Res. 1995; 55: 1373-1378PubMed Google Scholar). The role for CAe in facilitating CO2 removal has been demonstrated for CA4 in skeletal muscle (25Geers C. Gros G. Physiol. Rev. 2000; 80: 681-715Crossref PubMed Scopus (290) Google Scholar) and proposed for CA9 in tumors (2Swietach P. Vaughan-Jones R.D. Harris A.L. Cancer Metastasis Rev. 2007; 26: 299-310Crossref PubMed Scopus (426) Google Scholar, 26Svastová E. Hulíková A. Rafajová M. Zat'ovicová M. Gibadulinová A. Casini A. Cecchi A. Scozzafava A. Supuran C.T. Pastorek J. Pastoreková S. FEBS Lett. 2004; 577: 439-445Crossref PubMed Scopus (618) Google Scholar). Furthermore, CA9 expression is strongly up-regulated in hypoxia (5Wykoff C.C. Beasley N.J. Watson P.H. Turner K.J. Pastorek J. Sibtain A. Wilson G.D. Turley H. Talks K.L. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Harris A.L. Cancer Res. 2000; 60: 7075-7083PubMed Google Scholar), providing a mechanism by which CA9 levels are linked to diffusion distance. A consequence of facilitated CO2 removal is the attainment of a more uniformly alkaline pHi across the tissue. We demonstrated this recently in three-dimensional in vitro tissue models imaged for pHi (23Swietach P. Wigfield S. Cobden P. Supuran C.T. Harris A.L. Vaughan-Jones R.D. J. Biol. Chem. 2008; 283: 20473-20483Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). One prediction from that study is that CA9, although reducing pHi nonuniformity, will give rise to local extracellular acidity, particularly at the core of multicellular growths. If pHe is indeed acidified by CA9, the enzyme expression may be doubly beneficial for CO2-excreting tumors: it will help to attain (i) a favorable alkaline pHi for growth and (ii) an acidic pHe to facilitate invasiveness. Clinically, CA9 may serve as a target for drugs. In the present work, we image pHe using a novel, membrane-impermeant fluorescent pH dye in multicellular spheroid growths (∼35,000 cells) derived from the colon carcinoma cell line HCT116. We demonstrate a key role for CA9 in regulating both pHi and pHe. Furthermore, we show that, even in the hypoxic core of spheroids, the principal substrate for CA9 is cell-excreted CO2 and that the precise effect of CA9 on pHe depends on the relative efflux from cells of lactic acid versus CO2. The culture medium was Dulbecco's modified Eagle's medium containing 11 mmd-glucose or 2-deoxy-d-glucose (DOG) or d-galactose, buffered by 20 mm Hepes or 5% CO2, 22 mm HCO3−. The superfusate was normal Tyrode solutions containing 4.5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 11 mm glucose (or DOG or galactose), buffered by either 5% CO2 with 22 mm HCO3− (pH 7.4, 37 °C), 5% CO2 with 14 mm HCO3− (pH 7.2, 37 °C), or 1% CO2 with 2.2 mm HCO3− (pH 7.2, 37 °C) or a mixture of 2 mm + 2 mm, 10 mm + 10 mm, or 20 mm + 20 mm Hepes + Mes (pH 7.4, 37 °C). NaCl was added for a final osmolarity of 300 mOsm/kg. All of the superfusates were delivered at 2 ml/min and at 37 °C maintained by a feedback heater. Superfusates were changed rapidly ( 515 nm, ratioed, and calibrated with superfusates at different pH, buffered by 10 mm Hepes + 10 mm Mes. The CAe inhibitor 1-([4-sulfamoylphenyl)ethyl]-2,4,6-trimethyl pyridinium perchlorate (AP105) was synthesized in house and validated previously for inhibition potency (1Supuran C.T. Nat. Rev. Drug Discov. 2008; 7: 1-14Crossref PubMed Scopus (2540) Google Scholar). Acetazolamide (ATZ), a membrane-permeant CA inhibitor, was obtained from Sigma. HCT116 cells (radius 8.6 μm) were transfected with plasmid vector only (referred to as "empty vector" (or EV)) or plasmid vector with the cDNA for human CA9 (referred to as "CA9 expressor") and grown in McCoy's 5A medium (23Swietach P. Wigfield S. Cobden P. Supuran C.T. Harris A.L. Vaughan-Jones R.D. J. Biol. Chem. 2008; 283: 20473-20483Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). CA9 cDNA was a gift from Dr. J. Pastorek (Bratislava, Slovakia). Ice-cold cell suspension was homogenized 30–50 times and centrifuged at 3000 rpm for 10 min. Supernatant was microcentrifuged (Ti70 rotor; 8000 rpm, 45 min) to deposit the membrane fraction, which tested positive for Na+/K+ pump protein. Western blots tested for CA9 protein using mouse monoclonal M75 primary antibodies (a gift from Dr. J. Pastorek) (23Swietach P. Wigfield S. Cobden P. Supuran C.T. Harris A.L. Vaughan-Jones R.D. J. Biol. Chem. 2008; 283: 20473-20483Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The clone with the highest CA9 expression was selected for growing CA9-positive spheroids. CA9 expressor and EV HCT116 cells were cultured in McCoy's 5A medium. Aggregation into spheroids (28Sutherland R.M. Science. 1988; 240: 177-184Crossref PubMed Scopus (1534) Google Scholar) was initiated by plating 4 × 106 cells in 250-ml spinner flasks (Techne MCS, UK) spun at 40 rpm for 2–9 days. Unless stated otherwise, the media were buffered by 5% CO2, 22 mm HCO3− (pH 7.4 at 37 °C). Single cells were superfused in a chamber mounted on a Nikon Diaphot inverted microscope with a ×40 oil immersion objective. Excitation light alternating every 250 ms between 450 and 490 nm was provided by a xenon lamp monochromator. Fluorescence was collected by a photomultiplier tube at >515 nm. Confocal imaging offers high spatial resolution. Single cells or spheroids were imaged in a superfusion chamber mounted on a Leica IRBE microscope with a ×10 dry objective. The system was coupled to an normal Tyrode TCS confocal system with argon visible (514-, 458-, or 488-nm) and argon UV (351-nm) lasers. For single cell experiments, the pinhole was set to 1 Airy unit, and each cell was defined as a region of interest (ROI). For spheroid experiments, the Airy diameter in μm was set to 15% of spheroid diameter, a compromise between adequate confocality and good photon capture. The fluorescence images were used to identify the equatorial plane of the spheroid and to produce a spheroid outline. This outline was used to generate 10 concentric, nonoverlapping layered ROIs of width equal to a tenth of spheroid radius. ROI1 was defined as the ROI at the core, and ROI10 was defined as the peripheral ROI. The data are presented as the means ± S.E. and tested for statistical differences with t test (significant differences for p < 0.05). Fluorescein derivatives FS and DF were characterized for their fluorescent properties. Cell-free, 30 μm solutions of FS at different pH were studied in the confocal system under argon laser excitation, alternating between 458 and 488 nm every 4 s. To characterize DF, the cells were preloaded with dye at 10 μm for 10 min, and then dye-tagged cells were superfused with dye-free solutions at different pH under xenon lamp excitation, alternating between 450 and 490 nm every 0.25 s. Fig. 1A shows FS and DF emission at different pH. Fluorescence excited at the lower wavelength was less pH-sensitive, as it is nearer to the isosbestic point. The fluorescence excited at the two wavelengths was ratioed and plotted as a function of pHe in Fig. 1B. The pK values for FS and DF were estimated to be 6.4 and 7.6 (corrected for the ratio of maximum-to-minimum fluorescence at the lower wavelength, which offsets empirically estimated affinity (29Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar)). For a dye to report only pHe, it must not permeate cell membranes. The membrane permeability of FS was measured in EV cells, superfused with 30 μm dye, and imaged confocally. 45 min of exposure to FS increased cellular fluorescence by <3%, suggesting low membrane permeability (Fig. 1C). To determine whether DF inserts at the inner or outer leaflet of the membrane, DF-loaded cells were superfused with solutions that altered pHe alone (changing superfusate pHe) or pHi alone (superfusing with ammonium or acetate salts at constant pHe). DF fluorescence was only responsive to the former maneuver, indicating outer leaflet insertion. We also tested whether the dyes inhibit CA activity. A 10 nm solution of bovine red cell CA2, buffered by 20 mm Hepes, was tested for CA activity (23Swietach P. Wigfield S. Cobden P. Supuran C.T. Harris A.L. Vaughan-Jones R.D. J. Biol. Chem. 2008; 283: 20473-20483Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 0.5 ml of 100% CO2-saturated water was injected rapidly to 1.5 ml of enzyme suspension in a stirred chamber at 4 °C. Medium pH time courses were fitted with kinetic equations for CO2 hydration. The protocol was repeated in the presence of 30 μm FS and 100 μm ATZ. Catalysis by CA was inhibited by ATZ but unaffected by FS (Fig. 1D) or DF (not shown). Single HCT116 cells, transfected with vector alone, or vector with the CA9 gene, were assayed for CAe activity using the epifluorescence set-up. The cells were loaded with DF to report surface membrane pHe (Fig. 2, A and B) (27Stock C. Mueller M. Kraehling H. Mally S. Noël J. Eder C. Schwab A. Cell Physiol. Biochem. 2007; 20: 679-686Crossref PubMed Scopus (106) Google Scholar) and superfused with solutions buffered by 1% CO2, 2.2 mm HCO3− (pH 7.2). Superfusates of low buffering capacity optimized the amplitude of surface pHe changes. The superfusate was switched rapidly to one containing 30 mm ammonium (pH 7.2) for a period of 30 s. Exposure to an equilibrated solution of NH4+/NH3 drives rapid entry of NH3 into cells. Surface pHe is reduced as extracellular NH4+ deprotonates to replenish NH3 that has entered the cell. After ∼20 s, surface pHe re-equilibrates to 7.2. On subsequent removal of the extracellular weak base, NH3 is driven rapidly out of the cell, protonates at the outer cell surface, and transiently raises surface pHe. Because CO2/HCO3− buffer contributes to the release and consumption of H+ ions at the cell surface, the time courses of recorded pHe transients depend on CO2/HCO3− buffer kinetics, i.e. CAe activity. Fig. 2A (panel i) shows surface pHe transients measured in CA9-expressing cells in the absence and presence of the CAe inhibitor, AP105 (500 nm). Expression of CA9 protein in the membrane fraction was confirmed by Western blotting. The pHe transients were smaller and briefer in the absence of AP105. This was more evident in the alkaline direction; the area under the pHe transient was 57% larger in the presence of inhibitor. The smaller size of pHe transients recorded in the absence of drug is indicative of CAe activity. The same experimental protocol was performed on EV cells that lack CA9 protein in the membrane. In this case, the pHe transients (Fig. 2A, panel ii) were not affected by AP105, indicating the absence of CAe activity. To confirm the membrane impermeability of AP105 (and hence selectivity for CAe isoforms), we tested the inhibitory potency of AP105 (500 nm) on intracellular CA activity in EV cells and compared this with the potency of ATZ (100 μm), a membrane-permeant CAe inhibitor. EV cells were loaded with the pHi reporter dye carboxy-SNARF-1 (Fig. 2B) and superfused with dye-free solution. The superfusate was switched from one buffered by 20 mm Hepes (nominally CO2-free) to one buffered by 5% CO2, 22 mm HCO3−. Rapid solution change from 0 to 5% CO2 drives CO2 influx, intracellular hydration, and pHi acidification. The reverse solution change produces the opposite response (23Swietach P. Wigfield S. Cobden P. Supuran C.T. Harris A.L. Vaughan-Jones R.D. J. Biol. Chem. 2008; 283: 20473-20483Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Fig. 2B shows the pHi time course associated with these maneuvers. ATZ slowed the rate of pHi change, in both directions, but AP105 had no significant effect. The difference in response to the two drugs confirms that AP105 does not penetrate the cell membrane. The pHi time courses are in agreement with high membrane CO2 permeability (estimated to be ≥10 μm/s). The rate of pHi change upon the addition or removal of CO2 provides a measure of intracellular CO2/HCO3− buffering kinetics. Fig. 2C plots the initial rates of these pHi changes, normalized to the rate measured in ATZ. Catalysis by intracellular CA (CAi) isoforms alone (CAe blocked with AP105) is 4.0- and 3.4-fold in EV and CA9-expressing cells, respectively, i.e. slightly greater in EV cells. This finding is consistent with the inverse relationship between CA9 and CAi expression observed previously in RT112 cells (23Swietach P. Wigfield S. Cobden P. Supuran C.T. Harris A.L. Vaughan-Jones R.D. J. Biol. Chem. 2008; 283: 20473-20483Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Additional CAe activity in drug-free CA9-expressing cells augmented (by 15%) the kinetics of pHi change on removal of extracellular CO2, but not on its addition. CA9 has an asymmetric effect on CO2 hydration and HCO3− dehydration, because the spontaneous rate of the former is slower, making it more responsive to CA9 catalysis. The pHi time courses can also be used to derive intrinsic (non-CO2) buffering capacity (βi), which is given by the change in [HCO3−]i (which is equal to [HCO3−]o × 10pHi−pHo divided by the pHi change (23Swietach P. Wigfield S. Cobden P. Supuran C.T. Harris A.L. Vaughan-Jones R.D. J. Biol. Chem. 2008; 283: 20473-20483Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar)). βi was 27.3 ± 0.2 and 23.5 ± 0.2 mm/pH (n = 30) in CA9-expressing and EV cells, respectively (Fig. 2B). In summary, cells transfected with CA9 were positive for CA9 protein and displayed functional CAe activity. EV cells did not express CA9 protein and showed no CAe activity but a modestly higher CAi activity than CA9-expressing cells. The geometry of spheroids was characterized by confocal imaging with the membrane-permeant dye, 7-amino-4-methylcoumarin (AMC) (supplemental Fig. S1). Superfusion of spheroids with 10 μm AMC increased fluorescence throughout the s
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