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• W2024117007 abstract "Oligodendrocytes (OLs) are cells that produce myelin in the central nervous system. Here we use ratiometric pH indicator dye to analyze intracellular pH in OLs in culture. The results reveal alkaline microdomains, which predominate in the perikaryon and proximal dendrites, and acidic microdomains, which predominate in distal dendrites. Spatial nonuniformity of pH is generated by differential subcellular distribution of Na+/H+ exchanger (NHE), which is localized in a punctate distribution in the perikaryon and proximal processes, Na+/HCO3− cotransporter (NBC), which is localized in a punctate distribution in distal dendrites, and carbonic anhydrase isotype II (CAII), which is colocalized with either NHE or NBC. Inhibition of NHE activity by amiloride inhibits regeneration of alkaline microdomains after cytoplasmic acidification, whereas the inhibition of CAII activity with ethoxyzolamide inhibits acidification of dendrites. Fluorescence correlation spectroscopy analysis of CAII microinjected into OLs reveals freely diffusing protein throughout the cell as well as protein associated predominantly with NHE in the perikaryon and predominantly with NBC in the dendrites. Alkaline and acidic microdomains could be generated by transport metabolons consisting of CAII associated with NHE or NBC, respectively. This study provides the first evidence for pH microdomains in cells and describes a mechanism for how they are generated. Oligodendrocytes (OLs) are cells that produce myelin in the central nervous system. Here we use ratiometric pH indicator dye to analyze intracellular pH in OLs in culture. The results reveal alkaline microdomains, which predominate in the perikaryon and proximal dendrites, and acidic microdomains, which predominate in distal dendrites. Spatial nonuniformity of pH is generated by differential subcellular distribution of Na+/H+ exchanger (NHE), which is localized in a punctate distribution in the perikaryon and proximal processes, Na+/HCO3− cotransporter (NBC), which is localized in a punctate distribution in distal dendrites, and carbonic anhydrase isotype II (CAII), which is colocalized with either NHE or NBC. Inhibition of NHE activity by amiloride inhibits regeneration of alkaline microdomains after cytoplasmic acidification, whereas the inhibition of CAII activity with ethoxyzolamide inhibits acidification of dendrites. Fluorescence correlation spectroscopy analysis of CAII microinjected into OLs reveals freely diffusing protein throughout the cell as well as protein associated predominantly with NHE in the perikaryon and predominantly with NBC in the dendrites. Alkaline and acidic microdomains could be generated by transport metabolons consisting of CAII associated with NHE or NBC, respectively. This study provides the first evidence for pH microdomains in cells and describes a mechanism for how they are generated. Intracellular pH is an important modulator of cell function. Many enzymes exhibit pH dependence in the physiological range such that their activities are affected by small variations in intracellular pH. Until recently, intracellular pH was assumed to be spatially uniform because of fast diffusion of H+ ions and buffers. However, recent studies on cytoplasmic pH in epithelial cells, enterocytes, and myocytes indicate that H+ ions diffuse much more slowly in cytoplasm than in buffer (1Stewart A.K. Boyd C.A. Vaughan-Jones R.D. J. Physiol. 1999; 516: 209-217Crossref PubMed Scopus (70) Google Scholar, 2Joseph D. Tirmizi O. Zhang X.L. Crandall E.D. Lubman R.L. Am. J. Physiol. 2002; 282: L675-L683Google Scholar, 3Vaughan-Jones R.D. Peercy B.E. Keener J.P. Spitzer K.W. J. Physiol. 2002; 541: 139-158Crossref PubMed Scopus (77) Google Scholar), which means that localized generation or depletion of H+ ions within the cell can result in spatial nonuniformity of intracellular pH. This could cause intracellular variations in activities of pH-sensitive enzymes.Oligodendrocytes (OLs) 1The abbreviations used are: OL, oligodendrocyte; CA, carbonic anhydrase; CAII, carbonic anhydrase II; CNS, central nervous system; FCS, fluorescent correlation spectroscopy; NHE, sodium hydrogen exchanger; NBC, sodium bicarbonate cotransporter; SNAFL, seminaphthofluorescein; DiI, dialkylindocarbocyanine.1The abbreviations used are: OL, oligodendrocyte; CA, carbonic anhydrase; CAII, carbonic anhydrase II; CNS, central nervous system; FCS, fluorescent correlation spectroscopy; NHE, sodium hydrogen exchanger; NBC, sodium bicarbonate cotransporter; SNAFL, seminaphthofluorescein; DiI, dialkylindocarbocyanine. are glial cells that make spiral myelin sheaths around axons in the CNS. The large, flat, extended morphology of OLs in culture facilitates visualization of spatial nonuniformity of intracellular pH. In mature OLs, intracellular pH is predominantly determined by activities of three proteins: amiloride-sensitive Na+/H+ exchanger (NHE), ethoxyzolamide-sensitive carbonic anhydrase isotype II (CAII), and stilbene-insensitive Na+/HCO3− cotransporter (NBC) (4Boussouf A. Lambert R.C. Gaillard S. Glia. 1997; 19: 74-84Crossref PubMed Scopus (27) Google Scholar, 5Boussouf A. Gaillard S. J. Neurosci. Res. 2000; 59: 731-739Crossref PubMed Scopus (33) Google Scholar). A diisothiocyanatostilbene disulfonic acid-sensitive Cl−/HCO3− exchanger found in oligodendrocyte precursor cells is not present in mature oligodendrocytes (5Boussouf A. Gaillard S. J. Neurosci. Res. 2000; 59: 731-739Crossref PubMed Scopus (33) Google Scholar). Plasmalemmal NHE exchanges extracellular Na+ for intracellular H+ at 1:1 stoichiometry, depleting the cell of H+, thereby causing cellular alkalinization (6Yun C.H. Tse C.M. Nath S.K. Levine S.A. Brant S.R. Donowitz M. Am. J. Physiol. 1995; 269: G1-G11Crossref PubMed Scopus (38) Google Scholar, 7Noel J. Germain D. Vadnais J. Biochemistry. 2003; 42: 15361-15368Crossref PubMed Scopus (25) Google Scholar). Of the seven NHE isoforms, NHE1 appears to be the predominant form responsible for pH homeostasis in the CNS. Nonuniform distribution of NHE has been observed between the apical and basolateral membranes of proximal tubule epithelial cells (8Ives H.E. Yee V.J. Warnock D.G. J. Biol. Chem. 1983; 258: 13513-13516Abstract Full Text PDF PubMed Google Scholar), but the subcellular distribution of NHE in OLs has not been reported. In bicarbonate-free buffer, pHi recovery following an acid load requires external Na+ and is inhibited by the NHE inhibitor amiloride, indicating that under these conditions NHE is required for cellular alkalinization. Carbonic anhydrase (CA) catalyzes the conversion of CO2 and H2O into HCO3− and H+. Among 14 different CA isozymes, carbonic anhydrase II (CAII) is the predominant isozyme found in the brain, where it is concentrated in the myelin compartment of OLs (9Cammer W. Zhang H. Tansey F.A. J. Neurosci. Res. 1995; 40: 451-457Crossref PubMed Scopus (22) Google Scholar, 10Cammer W. Zhang H. J. Neuroimmunol. 1996; 67: 131-136Abstract Full Text PDF PubMed Scopus (17) Google Scholar, 11Cammer W.B. Brion L.P. Experientia Supplementa. 2000; 90: 475-489PubMed Google Scholar, 12Ridderstrale Y. Wistrand P.J. J. Neurocytol. 2000; 29: 263-269Crossref PubMed Scopus (13) Google Scholar). Electrogenic NBC exports Na+ and HCO3−, with a stoichiometry of 1:3 in mature OLs (4Boussouf A. Lambert R.C. Gaillard S. Glia. 1997; 19: 74-84Crossref PubMed Scopus (27) Google Scholar). Several NBC isoforms are expressed in mammalian CNS. A characteristic acidic cluster of amino acids (DNDD) in the cytoplasmic C-terminal region of NBC mediates association with the basic N-terminal region of CAII, creating a functional complex or “transport metabolon” to facilitate HCO3− export from the cell (13Sterling D. Reithmeier R.A. Casey J.R. J. Pancreas. 2001; 2: 165-170Google Scholar, 14Sterling D. Reithmeier R.A. Casey J.R. J. Biol. Chem. 2001; 276: 47886-47894Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 15Gross E. Pushkin A. Abuladze N. Fedotoff O. Kurtz I. J. Physiol. 2002; 544: 679-685Crossref PubMed Scopus (83) Google Scholar, 16Sterling D. Brown N.J. Supuran C.T. Casey J.R. Am. J. Physiol. 2002; 283: C1522-C1529Crossref PubMed Scopus (84) Google Scholar). Similar acidic regions in the cytoplasmic C-terminal region of NHE may also mediate interaction with CAII, creating a transport metabolon to facilitate H+ export, particularly under acidic conditions (17Li X. Alvarez B. Casey J.R. Reithmeier R.A. Fliegel L. J. Biol. Chem. 2002; 277: 36085-36091Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar).Here we show that OLs in culture contain spatially restricted regions of alkaline and acidic pH (pH microdomains) generated by differential subcellular distribution and colocalization of NHE, NBC, and CAII in transport metabolons. Alkaline microdomains predominate in the perikaryon and acidic microdomains predominate in the dendrites. This may provide a mechanism for differential regulation of pH-sensitive enzyme activities in different subcellular compartments.EXPERIMENTAL PROCEDURESCells and Reagents—Mouse or rat oligodendrocytes were isolated from mixed primary brain cell cultures and grown as previously described (18Ainger K. Avossa D. Diana A.S. Barry C. Barbarese E. Carson J.H. J. Cell Biol. 1997; 138: 1077-1087Crossref PubMed Scopus (256) Google Scholar). SNAFL-calcein acetoxymethyl ester, nigericin, N-isopropyl-N-methylamiloride hydrochloride, BODIPY FL amiloride, DiI, Texas Red dextran, Alexa Fluor monoclonal antibody labeling kit, and Alexa-labeled secondary antibodies were obtained from Molecular Probes, Inc. (Eugene, OR). Texas Red-conjugated and fluorescein-conjugated secondary antibodies were purchased from Jackson Immunoresearch (West Grove, PA). Ethoxyzolamide and CAII protein from bovine erythrocytes were obtained from Sigma. Rabbit anti-CAII antibody was provided by Dr. Wendy Cammer (Albert Einstein College of Medicine, Bronx, NY) or obtained from Chemicon (Pittsburgh, PA). Rabbit antibody raised against peptide 990-1035 of the rat kidney form of NBC-1 was purchased from Chemicon. Mouse monoclonal antibody against peptide 682-801 of rat NHE-1 isoform was obtained from BD Transduction Laboratories (Mississauga, Canada). Protein A-agarose beads and polyvinylidene difluoride membranes were from Bio-Rad. Enhanced chemiluminescence was from Pierce. Protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and pepstatin) were obtained from Sigma.Measurement of Intracellular pH—Intracellular pH was measured using cell-permeant SNAFL-calcein acetoxymethyl ester, a pH indicator with dual excitation maxima at 490/540 nm and dual emission maxima at 535/625 nm. The ratio of emission intensities at 625/535 nm for SNAFL-calcein is pH-dependent, increasing as pH increases from 6.4 to 7.6. To measure pHi in a single cell, OLs were loaded with 10 μm SNAFL-calcein acetoxymethyl ester for 30-40 min at room temperature in HEPES-buffered solution (25 mm HEPES, 140 mm NaCl, 5 mm KCl, 0.8 mm MgCl2·6H2O, 1.8 mm CaCl2, and 5.5 mm glucose, pH 7.3). After dye loading, the cells were rinsed twice to wash out unloaded dye and incubated for 15-20 min in HEPES buffer (pH 7.3). For calibration, KCl was substituted for NaCl in HEPES buffer, and pH was adjusted with KOH to 6.4, 6.7, 7.0, 7.3, and 7.6. Calibration measurements were done with dye-loaded cells treated with nigericin (a K+/H+ ionophore) to make cells permeable to H+ in solutions at five different pH values (19Thomas J.A. Buchsbaum R.N. Zimniak A. Racker E. Biochemistry. 1979; 18: 2210-2218Crossref PubMed Scopus (1762) Google Scholar, 20Boyarsky G. Ganz M.B. Sterzel R.B. Boron W.F Am. J. Physiol. 1988; 255: C844-C856Crossref PubMed Google Scholar). Using appropriate discriminatory filters, dual channel images were collected on Zeiss LSM 410 with a 63 × 1.4 numerical aperture oil immersion objective (Zeiss). Ratiometric images of 625/535 nm were generated for >5 cells at each pH, and the average ratio of intensities was calculated for each pH value. These intensity ratio values were used to generate a pH calibration curve for each experiment. The pHi values in OLs were determined by converting the intensity ratio values at each pixel to the corresponding pH value based on the calibration curve. The resulting ratiometric image was displayed in pseudocolor.Inhibitors—To examine the effects of NHE inhibitor on pHi, OLs were permeabilized with nigericin (10 μm) for 3-5 min and acidified in high K+ HEPES buffer (pH 6.4). The cells were then equilibrated in HEPES-buffered solution at pH 7.3 for 10 min in the presence or absence of the NHE inhibitor, N-isopropyl-N-methylamiloride hydrochloride (15 μm) to measure pHi recovery. To examine the effects of carbonic anhydrase inhibitor on pHi, OLs were treated with ethoxyzolamide (200 nm) for 5 min before imaging intracellular pH.Immunoprecipitation and Western Blotting—Oligodendrocytes from four 100-mm plates were lysed in 450 μl of buffer (150 mm NaCl, 50 mm Tris-HCl, pH 8.0, 1% (v/v) Nonidet P-40), containing a mixture of protease inhibitors (50 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin) and incubated at 4 °C for 1 h. Lysate was centrifuged (14,000 × g, 4 °C, 30 min). The supernatant was first precleared with 50 μl of washed Protein A-agarose beads by rocking at 4 °C for 1 h. The beads were then spun down, and the supernatant was split. For Western blotting, aliquots (10 μl) of total lysate were fractionated by SDS-PAGE and were immunoblotted by anti-CAII antibody, anti-NBC antibody, or anti-NHE1 antibody. Positive controls were 0.5 ng of CAII protein or rat brain microsomes. For co-immunoprecipitation, supernatants were incubated overnight at 4 °C with 10 μg of rabbit anti-CAII polyclonal antibodies or 2 μl of rabbit preimmune serum as a control. Protein A-agarose beads were then added and allowed to rock for 1.5 h. The beads were washed four times with 0.5 ml of lysis buffer and then boiled for 8 min in 50 μl of SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE on 10 or 8% polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes. After blocking the membrane with the TBS-T (140 mm NaCl, 20 mm Tris-HCl, 0.1% Tween 20, pH 7.6) containing 5% nonfat milk, Western blotting was performed with mouse anti-NHE monoclonal or rabbit anti-CAII polyclonal primary antibodies, followed by anti-mouse or anti-rabbit IgG coupled to horseradish peroxidase. Immunoreactive bands were visualized with enhanced chemiluminescence.Cell Staining, Confocal Microscopy, and Ratiometric Image Analysis—BODIPY-labeled amiloride (2 μm) and DiI (15 μg/ml) were used to label NHE and the cell membrane, respectively, in live cells. Rabbit anti-CAII antibody was used to stain CA in fixed cells. Texas Redconjugated dextran (Mr < 10,000), which diffuses uniformly throughout the cytoplasm and the nucleoplasm and thus serves as a cytoplasmic volume marker to normalize CAII concentrations in different regions, was microinjected into the cells prior to fixation. The NBC was stained using rabbit anti-NBC antibody and Alexa 488-labeled donkey anti-rabbit secondary antibody. Dual channel images were collected using a Zeiss LSM 410 confocal microscope with a 63 × 1.4 numerical aperture oil immersion objective with appropriate discriminatory filter sets. Neutral density attenuation was used to ensure that all regions of the image were within the dynamic range of the system. To determine the relative concentrations of NHE, NBC, and CAII in different subcellular compartments, ratios of intensities in the green and red channels (NHE/DiI, CAII/Texas Red dextran, and NBC/DiI) were calculated and displayed in pseudocolor using NIH Image software, version 1.62 (National Institutes of Health).Colocalization Analysis—For CAII·NHE double labeling, rabbit polyclonal anti-CAII and mouse monoclonal anti-NHE primary antibodies were used, followed by Texas Red-labeled donkey anti-rabbit and Alexa 488-labeled donkey anti-mouse secondary antibodies. For CAII·NBC double labeling, CAII protein (100 μg/ml) was labeled with Alexa 647 using an Alexa Fluor monoclonal antibody labeling kit from Molecular Probes. Alexa 647-labeled CAII (10 μg/ml) was microinjected into the OLs followed by fixation with 3.7% formaldehyde. NBC was stained as above. Dual channel images were collected using a Zeiss LSM 510 confocal microscope with a 63 × 1.4 numerical aperture oil immersion objective with appropriate discriminatory filter sets. To analyze individual puncta, uniform staining was subtracted in both green and red channels using Photoshop (Adobe). Several hundred individual, well resolved puncta were selected in both channels, and the intensities of CAII and NHE or NBC associated with each punctum were measured as described previously (21King S.M. Barbarese E. Dillman III, J.F. Patel-King R.S. Carson J.H. Pfister K.K. J. Biol. Chem. 1996; 271: 19358-19366Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) and plotted in a scatter diagram.Fluorescence Correlation Spectroscopy Analysis—To measure intracellular concentrations and diffusion times for CAII in OLs, CAII protein was fluorescently labeled with Alexa 647, using the Alexa Fluor monoclonal antibody labeling kit from Molecular Probes, and microinjected into OLs. The fluorescence correlation spectroscopy (FCS) observation volume was positioned in either the perikaryon or dendrites. In our FCS system (Zeiss Confocor II), confocal optics is used to define the observation volume (<1 fl) illuminated by a stationary laser beam. Fluorescence fluctuation within the observation volume is detected using low noise avalanche photodiode detectors with single photon sensitivity. Photons in the observation volume were recorded over 5 s, and the measurement was repeated five times at each position in the cell. The dynamic properties of CAII molecules at each subcellular location were analyzed by applying the autocorrelation method to the entire fluctuation record (22Magde D. Elson E. Webb W.W. Phys. Rev. Lett. 1972; 29: 705-708Crossref Scopus (1447) Google Scholar). The autocorrelation function is calculated by comparing the fluorescence intensity at time t to the intensity at time t + τ. The autocorrelation function for each measurement was computed using Zeiss Confocor software. Autocorrelation curves with significant photobleaching were discarded, and the remaining curves for each subcellular location were averaged. The averaged autocorrelation curves were globally fit into the theoretical autocorrelation function for three-dimensional diffusion.G(t)=(1+ytripexp(−t/τtrip)1−ytrip)1N∑i=1CNyi(1+tτi)1+ω2tτi(Eq. 1) Where N represents the number of particles, ytrip is triplet state fraction, τtrip is triplet state time, CN is the total number of diffusion components, yi is the fraction of the diffusion component i, τi is diffusion time for component i, and ω is the structural parameter of the observation volume.Manipulation of raw FCS data and nonlinear least squares curve fitting was performed using a system of specialized MATLAB scripts (available upon request). Reciprocals of the S.D. values for each point of the experimental autocorrelation curve were used as weights for fitting.If fluorescent CAII aggregates or binds to other molecules and if aggregation or binding alters its diffusion properties, the fraction bound and fraction unbound can be determined. This provides a measure of the diffusion times and proportions for each component. The proportions of each component were converted to absolute concentrations as follows. The total number of endogenous CAII molecules in the cell (∼10,000 molecules/cell) was determined by quantitative Western blotting (data not shown). The overall concentration of endogenous CAII in the entire cell was estimated based on the total number of molecules per cell and the total volume of the OL (estimated to be ∼1 pl). The volume fraction of the perikaryon and proximal dendrites was estimated to be approximately equal to the volume fraction of the distal dendrites based on their relative areas and the estimated average thickness of cytoplasm in the two compartments. The average relative concentration of CAII in the perikaryon and proximal dendrites was approximately half that in the distal dendrites, based on quantitative immunofluorescence. This allows estimation of the average absolute concentration of endogenous CAII in the perikaryon and proximal dendrites and in the distal dendrites. Fitting the FCS autocorrelation data provides a measure of the relative proportions of microinjected fluorescent CAII with different autocorrelation times. Assuming that FCS autocorrelations times for exogenous fluorescent CAII reflect the distribution of endogenous unlabeled CAII, the absolute concentrations of each component in each subcellular compartment can be estimated.RESULTSpH Microdomains in OLs—OLs were incubated with ratiometric pH indicator dye (seminaphthofluorescein (SNAFL)-calcein acetoxymethyl ester) in bicarbonate-free HEPES buffer (pH 7.3). Following dye loading, dual channel confocal images were collected and converted to ratiometric images to visualize pHi. Calibration was performed with cells permeabilized with nigericin (a K+/H+ ionophore) in high K+ buffer at five different pH values (pH 6.4, 6.7, 7.0, 7.3, 7.6). Nigericin-treated cells showed a uniform distribution of pH throughout the cytoplasm (Fig. 1A shows representative cells at pH 6.4, 6.7, 7.0, 7.3, and 7.6). Ratiometric values were measured in five cells at each pH to generate a calibration curve for intracellular pH (Fig. 1A). Fig. 1B shows a representative dual channel image of an OL. The ratios between the channels are not uniform throughout the cell; some regions appear more reddish and some appear more greenish, indicating spatial nonuniformity of pH. The calibration data from Fig. 1A was used to calculate intracellular pH based on the ratios between the two channels at each position in the cell. Fig. 1C shows a ratiometric image of the cell in Fig. 1B, where intracellular pH is represented in pseudocolor. The perikaryon and some dendrites appear more alkaline (red), whereas other dendrites appear more acidic (blue). Close examination of the image (Fig. 1C, inset) reveals numerous alkaline microdomains (red) and acidic microdomains (blue) scattered throughout the cell in close apposition to the cell membrane. Microdomains are defined as discrete regions (generally ∼1 μm in diameter) with pH values that differ from the surrounding cytoplasm by >0.1 pH unit. In the perikaryon and in some dendrites, alkaline microdomains predominate, making the overall intracellular pH in these regions appear more alkaline. In other dendrites, acidic microdomains predominate, making the overall pH in these regions appear more acidic. Alkaline microdomains may reflect regions of local proton depletion, and acidic microdomains may represent regions of local proton generation. Spatial nonuniformity in the ratiometric image shown in Fig. 1C is interpreted as evidence for nonuniform intracellular pH. The relatively uniform intracellular pH in nigericin-permeabilized cells used for calibration (Fig. 1A) indicates that spatial nonuniformity of pH in intact cells is not due to nonuniform intracellular dye binding, noise in the imaging system, or imaging artifacts.Subcellular Distribution of NHE, CAII, and NBC in OLs— The function of NHE is to export protons from the cytoplasm. Fig. 2A shows that NHE-1 is detected in OLs by Western blotting. To analyze the subcellular distribution of NHE, OLs were incubated with BODIPY-amiloride, which binds to NHE, and with DiI, a lipophilic carbocyanine dye, which labels the plasma membrane uniformly. Ratiometric images displaying NHE intensity divided by DiI intensity at each pixel provide a measure of the relative concentration of NHE at each point on the cell membrane. NHE is concentrated in a punctate distribution in the perikaryon and proximal processes. Each punctum presumably represents a cluster of NHE molecules formed by association of individual NHE molecules within the plane of the membrane. Since the function of NHE is to exchange extracellular Na+ for intracellular H+, punctate distribution of NHE may result in local depletion of H+, which could create alkaline microdomains in the vicinity of the clusters.Fig. 2NHE, CA, and NBC in OLs. A-D provide evidence for NHE in OLs. A shows a Western blot of NHE-1 in OL lysate. The control lane (C) was loaded with lysate from rat liver microsomes. The positions of molecular weight standards are indicated. B-D show the subcellular distribution of NHE-1. OLs were incubated with DiI (15 μg/ml) for 20 min at room temperature to label the cell membrane. BODIPY-labeled amiloride (2 μm) was used to label NHE protein in OLs by incubating for 10 min. The green channel (B) shows amiloride labeling of NHE, and the red channel (C) shows the DiI labeling of the plasma membrane. The single channel images are displayed in inverted grayscale to facilitate visualization of fine processes. The relative concentration of NHE at each pixel was normalized by dividing the intensity in the NHE image by the intensity in the corresponding pixel in the DiI image. The ratiometric image of NHE/DiI (D) is displayed in pseudocolor, where the highest NHE concentration is displayed in red and the lowest in blue. E-H provide evidence for CAII in OLs. E shows Western blotting of CAII in OL lysate. The control lane was loaded with purified CAII. F-H show the subcellular distribution of CAII in OLs. Texas Red-conjugated dextran (Mr <10,000) was microinjected into the cells as a cytoplasmic volume marker, followed by fixation with 3.7% formaldehyde and immunolabeling with rabbit anti-CAII primary antibodies and fluorescein-labeled donkey anti-rabbit secondary antibodies. CAII and cytoplasmic volume were visualized in green (F) and red (G) channels, respectively, and are displayed in inverted grayscale. The intensity of CAII in the green channel was divided by the intensity of Texas Red dextran in the red channel. The ratiometric image (H) of CAII/cytoplasmic volume is displayed in pseudocolor, where the highest CAII concentration is shown in blue and the lowest in red. I-L provide evidence for NBC in OLs. I shows Western blotting of NBC-1 in OL lysate. The control lane was loaded with lysate from rat liver microsomes. J-L show the subcellular distribution of NBC-1 in OLs. Cells were fixed and stained with DiI to label the plasma membrane, and NBC was immunolabeled with rabbit anti-NBC primary antibody and Alexa 488-labeled anti-rabbit secondary antibody. J and K show the staining of NBC and plasma membrane, respectively, displayed in inverted grayscale. The ratiometric image (L) of NBC/DiI is displayed in pseudocolor, where the highest NBC concentration is shown in blue and the lowest in red. Note that the pseudocolor coding in H and L is inverted relative to D to facilitate visualization of color variations in the distal dendrites.View Large Image Figure ViewerDownload (PPT)CAII reversibly converts H2O and CO2 to H+ and HCO3−. We have determined, by quantitative Western blotting (Fig. 2B), that OLs contain ∼10,000 CAII molecules/cell (data not shown). To analyze the subcellular distribution of CAII, OLs were microinjected with Texas Red-labeled dextran as a cytoplasmic volume marker and then fixed and stained with antibody to CAII. The CAII intensity divided by the Texas Red dextran intensity, displayed as a ratiometric image in pseudocolor, provides an indication of the relative concentration of CAII at each position in the cell. CAII is distributed at low intensity uniformly throughout the cell and at higher intensity in a discrete punctate distribution in the dendrites. Based on the number of puncta and the number of CAII molecules per cell, each punctum represents 10-100 CAII molecules. The overall concentration of CAII is higher in the dendrites than in the perikaryon, with different concentrations in individual processes.NBC is a cotransporter that exports intracellular Na+ and HCO3− from the cell, suggesting that it may play a role in removing HCO3− generated by CAII in the periphery. Fig. 2C shows that NBC-1 is detected in OLs by Western blotting. To examine the distribution of NBC in OLs, cells were incubated with DiI to label the plasma membrane uniformly and then immunostained with antibody to NBC. The ratiometric image of NBC/DiI displaying the intensity of NBC divided by DiI intensity in pseudocolor provides a measure of NBC concentration at each position on the membrane. NBC appears to be localized in a punctate distribution in most dendrites. Each punctum presumably represents a cluster of NBC molecules formed by association of individual NBC molecules in the plane of the bilayer. Since the function of NBC is to export intracellular HCO3−, the punctate distribution of NBC may result in local accumulation of H+, which could create acidic microdomains in the vicinity of the clusters.Inhibition of NHE or CAII Affects Intracellular pH in OLs—To determine whether NHE activity regulates intracellular pH, OLs loaded with SNAFL-calcein were acidified by nigericin treatment at pH 6.4, followed by equilibration in buffer at pH 7.3, in the absence or presence of amiloride to inhibit NHE function (Fig. 3, A and B). Amiloride treatment inhibited overall pH recovery and also inhibited regeneration of alkaline microdomains, indicating that NHE contributes to establishing and maintaining alkaline intracellular pH in OLs. Since amiloride is known to inhibit several different sodium/proton antiporter isoforms (23Smart S.C. Locurto A. el Schultz J. Sagar K.B. Warltier D.C. J. Am. Coll. Cardiol. 1995; 26: 1365-1373Cros" @default.
• W2024117007 created "2016-06-24" @default.
• W2024117007 creator A5035634241 @default.
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• W2024117007 date "2004-08-01" @default.
• W2024117007 modified "2023-10-18" @default.
• W2024117007 title "pH Microdomains in Oligodendrocytes" @default.
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• W2024117007 doi "https://doi.org/10.1074/jbc.m403099200" @default.
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