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Immunofluorescence revealed the presence of NHE-1 in the nuclear membranes of rat cardiomyocytes and isolated nuclei of human, rabbit, and rat aortic and liver tissues Ghassan Bkaily, Moni Nader, Levon Avedanian, Danielle Jacques, Claudine Perrault, Dima Abdel-Samad, Pedro D’Orléans-Juste, Fernand Gobeil, and Khaled M. Hazzouri

Abstract: Using immunofluorescence and 3-dimensional confocal microscopy techniques, the present study was designed to verify if NHE-1 is present at the level of the nuclear membrane in cells that are known to express this type of exchanger. Nuclei were isolated from aortic tissues of adult human, rabbit, and rats, as well as from liver tissues of human fetus, and adult rabbit and rat. In addition, cultured ventricular cardiomyocytes were isolated from 2-week-old rat. Our results showed the presence of NHE-1 in isolated nuclei of aortic vascular smooth muscle and liver of human, rabbit, and rat. NHE-1 seems to be distributed throughout the isolated nucleus and more particularly at the level of the nuclear membranes. The relative fluorescence density of NHE-1 was significantly higher (p < 0.05) in isolated liver nuclei of human, when compared with those of rabbit and rat. However, in isolated nuclei of aortic vascular smooth muscle, the relative fluorescence density of NHE-1 was significantly (p < 0.001) higher in the rabbit when compared with human and rat. In cultured rat ventricular cardiomyocytes, NHE-1 fluorescent labeling could be easily seen throughout the cell, including the nucleus, and more particularly at both the sarcolemma and the nuclear membranes. In rat cardiomyocytes, the relative fluorescence density of NHE-1 of the sarcolemma membrane, including the cytosol, was significantly lower than that of the whole nucleus (including the nuclear envelope membranes). In conclusion, our results showed that NHE-1 is present at the nuclear membranes and in the nucleoplasm and its distribution and density may depend on cell type and species used. These results suggest that nuclear membranes’ NHE-1 may play a role in the modulation of intranuclear pH. Key words: NHE-1, heart, aorta, liver, nuclear membranes, nucleus.

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Résumé : La présente étude a été conçue pour vérifier la présence de NHE-1 au niveau des membranes nucléaires des cellules qui sont connues pour exprimer ce type d’échangeur. Pour ce faire, nous avons utilisé les techniques de microscopie confocale tridimensionnelle et d’immunofluorescence. Les noyaux ont été isolés des tissus aortiques d’humains, de lapins et de rats adultes ainsi que des tissus hépatiques de fœtus humains, de rats et de lapins adultes. De plus, des cardiomyocytes ventriculaires cultivés de rats âgés de 2 semaines ont été isolés. Nos résultats ont montré la présence de NHE-1 dans les noyaux isolés du muscle lisse vasculaire aortique et du foie des humains, des lapins et des rats. NHE-1 semble être distribué dans tout le noyau isolé et plus particulièrement au niveau des membranes nucléaires. Dans les noyaux hépatiques isolés, la densité de fluorescence relative de NHE-1 a été significativement plus élevée (p < 0,05) chez les humains que chez les lapins et les rats. Toutefois, dans les noyaux isolés du muscle lisse aortique, elle a été significativement plus élevée (p < 0,001) chez les lapins. Dans les cardiomyocytes ventriculaires cultivés de rats, le marquage fluorescent de NHE-1 a pu être observé dans toute la cellule, incluant le noyau, et plus particulièrement au niveau des membranes sarcolemmiques et nucléaires. Dans les cardiomyocytes de rats, la densité

Received 18 July 2004. Accepted 8 September 2004. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 20 October 2004. G. Bkaily,1 M. Nader, L. Avedanian, D. Jacques, C. Perrault, D. Abdel-Samad, and K.M. Hazzouri. Department of Anatomy and Cell Biology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, 3001– 12th Avenue North, QC J1H 5N4, Canada. P. D’Orléans-Juste and F. Gobeil. Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QC J1H 5N4, Canada. 1

Corresponding author (e-mail: [email protected]).

Can. J. Physiol. Pharmacol. 82: 805–811 (2004)

doi: 10.1139/Y04-119

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Can. J. Physiol. Pharmacol. Vol. 82, 2004 fluorescente relative de NHE-1 au niveau de la membrane sarcolemmique, incluant le cytosol, a été significativement plus faible que celle du noyau entier (incluant les membranes nucléaires). En conclusion, nos résultats ont montré que NHE-1 était présent au niveau des membranes nucléaires et dans le nucléoplasme, et que sa distribution et sa densité pourraient dépendre du type de cellules et de l’espèce utilisée. Ces résultats donnent à penser que le NHE-1 des membranes nucléaires pourrait jouer un rôle dans la modulation du pH intranucléaire. Mots clés : NHE-1, cœur, aorte, foie, membranes nucléaires, noyau. [Traduit par la Rédaction]

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Introduction Growing evidence has shown that certain channels (Bkaily 1994; Mazzanti et al. 2001), Ca2+ pump (Abernica et al. 2003; Carafoli et al. 1997), Na+-K+ pump (Garner 2002), Na+-Ca2+ exchanger (Xie et al. 2002) as well as certain types of receptors (Bkaily et al. 1997, 2003a, 2003b; Jacques et al. 2003a, 2003b; Gobeil et al. 2002, 2003) are present at the level of the nuclear envelope membranes as well as within the nucleus of several types of cells of different species. Furthermore, protein synthesis also seems to take place in the nucleus (Iborra et al. 2001; Bkaily et al. 2003b). Recent work from our laboratory showed that extracellular changes in pH induced both cytosolic and nucleoplasmic pH changes in human aortic vascular smooth muscle cells (VSMCs), and it was suggested that these nucleoplasmic pH changes could be due to the presence of the Na+-H+ exchanger at the nuclear envelope membranes (Bkaily et al. 1997). To the present date, NHE-1 was only localized at the cell membrane of several cell types such as cardiomyocytes (Karmazyn 2001; Karmazyn et al. 2003), VSMCs, and hepatocytes. However, there is no information whether, like the Na+-Ca2+ exchanger (Xie et al. 2002) and the Na+-K+ pump (Garner 2002), NHE1 is also present at the nuclear envelope membranes of VSMCs, cardiomyocytes, and hepatocytes. In the present work, we used immunofluorescent labeling of NHE-1 and 3dimensional (3-D) confocal microscopy to determine if NHE-1 is present in isolated nuclei of VSMCs and hepatocytes as well as in intact single cardiomyocytes, and whether the nuclear distribution and the relative density of the exchanger depend on cell type and (or) animal species.

Materials and methods Isolation of the nuclei Intact nuclei of VSMCs were isolated from healthy adult human donors as well as from adult rat and rabbit aortas (after removing the connective tissue and the endothelium). Intact nuclei of hepatocytes were isolated from livers of human fetal (20 week-old) donors as well as adult rats and rabbits. The work was performed in accordance with requirements of the Institutional Review Committee for the use of human and animal material. Quebec Transplant supplied the donors’ aortas. The animals were supplied by Charles River Canada Inc. (St. Constant, Que.) and were cared for in accordance with guidelines of the Canadian Council of Animal Care (Ottawa, Ont.). The pure, intact nuclei were isolated as previously described (Gobeil et al. 2002) with some modifications. In brief, after homogenization with a dounce tissue grinder (tight pestle; Belloco

Glass, VWR, Mount-Royal, Que.), the homogenate was centrifuged at 800 g for 10 min at 4 °C. For nuclei from liver cells, isolation was carried out by ultracentrifugation through a sucrose gradient (Gobeil et al. 2002). However, the nuclei of VSMCs were only filtered through a gauze and washed 3 times at 800 g (5 min each) at 5 °C. For both preparations, the pellet of nuclei was resuspended in a buffer solution (23 mmol/L HEPES, 125 mmol/L KCl, 2 mmol/L K2HP04, 4 mmol/L MgCl2 and 400 nmol/L CaCl2, pH 7.2). The morphological integrity and purity (98%) were assessed by light microscopy, after trypan blue staining. In addition, purity was also assessed using Syto-11 live cell nucleic acid stain (Molecular Probes, Eugene, Ore). Cell culture Two week-old rat cardiomyocytes were isolated from lower third of the ventricle and cultured as previously described (Bkaily et al. 1997). In brief, after isolation, the cardiomyocytes were cultured in Hanks Minimum Essential Medium (HMEM, Gibco InVitrogen Canada Inc., Burlington, Ont.) supplemented with 10% FBS (GIBCOBRL, Burlington, Ont.). Immunofluorescence The method of immunofluorescence for both nuclei and intact single cells is the same as was described previously (Bkaily et al. 2003a, 2003b). In brief, the cells or the isolated nuclei were fixed for 10 min in ice cold 4% paraformaldehyde (Brismar et al. 1998). After washing with phosphate buffer saline (PBS), nuclei or cells were incubated for 10 min with PBS containing sodium borohydride (2 mg/mL), then permeabilized and blocked with 0.1% Triton X-100, 7% normal rabbit serum (NRS), and 5% non-fat dry milk (NFDM) for 30 min. Finally, the nuclei or the cells were washed twice in PBS and incubated overnight at 4 °C with primary goat anti-NHE-1 polyclonal antibody, SC16097 (Santa Cruz Biotechnologies, Calif.), in PBS containing 1.4% NRS, 1% NFDM, and 0.1% Triton X-100. After 2 PBS washes, the nuclei or the cells were incubated for 1 h at room temperature with secondary rabbit anti-goat-IgG-Alexa Fluor 488 antibody for NHE-1 immunofluorescence. The nuclei or the cells were examined using 3-D confocal microscopy. The use of 3-D imaging and fluorescence intensity measurement of the isolated nucleus permits us to ensure the quality of the intact isolated nucleus and to avoid contamination of the measurement with fluorescence labelling of cell fractions that are, most of the time, very difficult to completely eliminate from the pellet suspension of isolated nuclei. The PBS is composed of 8.0 g NaCl, 0.2 g KCl, 1.4 g © 2004 NRC Canada

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anhydrous Na2HPO4, and 0.2 g KH2PO4 dissolved in 1 L of distilled water. Confocal microscopy Isolated nuclei or cells were examined with a Multiprobe 2001 confocal argon laser scanning system (CLSM) (Molecular Dynamics, Sunnyvale, Calif.) equipped with a Nikon Diaphot epifluorescence inverted microscope and a 60× (1.4 NA) Nikon Oil Plan achromat objective (Bkaily et al. 1997, 2003a, 2003b). The 488-nm argon laser line (9.0 mV) was directed to the sample via a 510 nm primary dichroic filter and attenuated with a 3% neutral density filter to reduce photobleaching (Bkaily et al. 1997). Pinhole size was set at 100 µm. The image size was 512 × 512 pixels with a pixel size of 0.34 µm. Laser line intensity, photometric gain, PMT settings, and filter attenuations were kept constant throughout the experimental procedures (Bkaily et al. 1997, 2003b). Measurements of fluorescence were performed from 3-D reconstructions of the nuclei or the cells (including the nucleus) as described previously (Bkaily et al. 1997, 2003a, 2003b). In brief, after acquiring a series of consecutive images following optical Z-sectioning of a nucleus or a cell, 3D imaging was performed. The intensities were measured throughout the entire volume of the sections of each compartment (cytosol or nucleus) to provide 3-D information. We then calculated the volume intensity of the measured nucleus or cell compartments using the 3-D Imaging Software, Image Space (Molecular Dynamics). Volume integration over the various compartments would give an estimate of the total amount of the exchangers in each of the 2 compartments. Since our measurements are done in the whole volume of a compartment and not in 1 or few sections of the volume of the nucleus or compartment of the cell, our whole volume measurements eliminate any possible problem concerning drifting of the Z-line. The 3-D images were presented as top view projections (horizontal) or as volumetric projection 3-D images with a 45° rotation around the Y-axis using the software, Image Space (Molecular Dynamics). The top view 3-D image permits a “look-through” of the whole nucleus or the cell, but because of the low thickness of the nucleus and the cell, the top view presentation does not permit a clear view of NHE-1 located at the level of the nuclear envelope membranes or the sarcolemma. However, the lateral view with a 45° rotation permits a better view of the isolated nucleus or cell membrane NHE-1. Since the nuclear envelope membranes are very thick, both types of presentation permit a clear view of the nuclear membranes. The nuclear envelope membranes’ fluorescence of NHE-1 was isolated from that of the whole nucleus by subtracting the fluorescence labeling of the nucleoplasm, marked by Syto11, from the whole nuclear NHE-1 fluorescence labeling. Nuclear staining and perinuclear envelope membrane demarcation At the end of each experiment the nucleus was stained with 100 nmol/L of live cell nucleic acid stain Syto-11 (Molecular Probes). Syto-11 stain is effective in staining the nucleus of a wide range of cell types, including cultured mammalian cells (Bkaily et al. 1997). The Syto-11 staining was used to delineate nuclear from cytosolic fluorescence as

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well as the nuclear envelope membranes from the nucleoplasm (Bkaily et al. 1997). Serial optical scans were taken immediately after development of the stain (~8–10 min) while maintaining positioning, number of sections, and step size constant throughout the experimental procedures. Three-dimensional reconstructions of the nucleus (Bkaily et al. 1997) were performed as described previously (Bkaily et al. 1997) in the section on volume rendering and nuclear fluorescence measurements and were used as templates to delineate nuclear from cytosolic fluorescence. In brief, for nuclear envelope membranes’ digital imaging separation, the nucleoplasmic area, following syto-11 staining, was isolated from the rest of the nucleus by setting a lower intensity threshold filter to confine relevant pixels. Hence, after removing the nucleoplasm from the surrounding nuclear envelope membranes, we were able to delimit the perinuclear envelope membranes entirely. Materials All other materials, if not stated otherwise, were purchased from Sigma-Aldrich (St. Louis, Mo.). Statistics All results are expressed as mean values ± SEM where n is the number of different experiments. Each experiment represents the mean of measured nuclei from each tissue that were within the field of the microscope and that showed Syto-11 labeling (3 to 22 nuclei). When applicable, statistical significance was determined using the Student t test provided by the program Graph Pad™ (V2.4a). p values < 0.05 were considered significant. Results Presence of NHE-1 in isolated nuclei of aortic VSMCs from human, rabbit, and rat By using specific antibodies against NHE-1, we observed the presence of this type of Na+-H+ exchanger in isolated intact nuclei of aortas from human, rabbit, and rat. Figure 1 shows examples and Fig. 4a summarizes the results. The absence of fluorescence in the negative controls (primary antibody in the presence of the peptide sequence used to generate the antibody) demonstrates the labeling specificity of the antibody used in our experiments (Figs. 1d, 1i, and 1n). As can be seen in Fig. 1, the NHE-1 is distributed in a non-homogeneous way all through the nucleus of human (Fig. 1a), rabbit (Fig. 1f), and rat (Fig. 1k) (top view, lookthrough images) with higher fluorescence density at the level of nuclear membranes, as demonstrated by the nuclear envelope 3-D images, obtained by subtracting the nucleoplasmic volume (syto-11 staining, Figs. 1b, 1g, and 1l) from that of the whole nucleus (Figs. 1a, 1f, and 1k respectively). However, in rabbit nuclei, the level of fluorescence density of NHE-1 seems to be higher than those of both human and rat whole nuclei. Figure 4a shows that the mean fluorescence intensity labeling of the NHE-1 was similar in isolated nuclei of human (5.96 ± 0.69, n = 3 aortas from different donors) and rat (5.55 ± 0.10, n = 4). However, the fluorescence intensity labeling of the NHE-1 in isolated nuclei from rab© 2004 NRC Canada

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Fig. 1. Indirect immunofluorescence and 3-D reconstruction top-view, look-through images of NHE-1 fluorescence intensity and distribution in isolated nuclei from aortas of adult human (a), rabbit (f), and rat (k) and their respective nucleoplasmic Syto-11 labeling ((b), (g), and (l)). Panels (c), (h), and (m) are respectively the NHE-1 fluorescence labeling of the NHE-1 in nuclear envelope membranes of isolated whole nuclei from human (panel (a)), rabbit (panel (f)), and rat (panel k) obtained after subtraction of the nucleoplasmic Syto11 labeling ((b), (g), and (l) respectively) from the whole nucleus ((a), (f), and (k) respectively). Panels (d), (i), and (n) are the negative controls in the presence of blocking peptide and in the absence of the primary antibody. Panels (e), (j), and (o) are respectively the Syto-11 staining of the experiment in panels (d), (i), and (n). The pseudocolour scale represents NHE-1 immunofluorescence level from 0 (black colour) to 255 (white colour). The white bar is 5 µm. Note the elongated shape of the isolated nuclei, which is the same as those normally reported in intact tissues.

bit (17.68 ± 0.86, n = 5) was significantly (p < 0.001) higher than those found in human and rat nuclei.

rabbit (23.28 ± 0.79, n = 6) and rat (21.53 ± 0.15, n = 5) (Fig. 4b).

Presence of NHE-1 in isolated liver nuclei from human, rabbit, and rat Similar to the nuclei of VSMCs, the nuclei of human, rabbit, and rat liver cells showed the presence of NHE-1. Figure 2 shows examples and Fig. 4b summarizes the results. The distribution of the exchanger in isolated nuclei of liver tissues from human (Fig. 2a), rabbit (Fig. 2f), and rat (Fig. 2k) was non-homogeneous and high cluster-like labeling could be seen at the level of the nuclear membranes, as well as the nucleoplasm (Fig. 2). Subtraction of the nucleoplasm (syto-11 labeling, Figs. 2b, 2g, and 2l) from the whole nucleus (Figs. 2a, 2f, and 2k respectively) permits the 3-D view of the nuclear envelope membranes of human (Fig. 2c), rabbit (Fig. 2h), and rat (Fig. 2m) liver tissues. As can be seen in panels of Fig. 2b, the NHE-1 seems to be distributed non-homogeneously in the nuclear envelope membranes. The fluorescent intensity labeling of the exchanger was significantly (p < 0.05) higher in isolated nuclei from human (34.01 ± 6.61, n = 3), as compared with those from

Presence of NHE-1 in the nuclear membranes of intact cardiomyocytes from 2 week-old rats Single heart cells of young rats showed a nonhomogeneous distribution of NHE-1 all through the cell with a very high level and clear fluorescent labeling of the exchanger at both the sarcolemma and the nuclear envelope membranes (Fig. 3a). To better see the nucleus, the middle portion of the cell in the lower part of Fig. 3a has been sectioned (Bkaily 1994; Bkaily et al. 1997) and clearly showed the presence of NHE-1 in the nuclear envelope membranes (Fig. 3b). Labeling of NHE-1 could be also seen at the level of the nucleoplasm and, to a lesser extent, at the cytoplasmic level. Fluorescence density of NHE-1 labeling was separately measured at the levels of the cytosol (including the sarcolemma membrane) and the whole nucleus (including the nuclear envelope membranes) (Bkaily et al. 1997, 2003a, 2003b). These 3-D volume measurements showed that the density of NHE-1 in the whole nucleus (including the nuclear envelope membranes) was significantly (p < 0.001) © 2004 NRC Canada

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Fig. 2. Indirect immunofluorescence and 3-D reconstruction top-view, look-through images of NHE-1 fluorescence intensity and distribution in isolated nuclei from livers of 20 week-old fetal human (a), adult rabbit (f), and adult rat (k) and their respective nucleoplasmic Syto-11 labeling ((b), (g), and (l)). Panels c, h, and m are respectively the NHE-1 fluorescence labeling of the NHE-1 in nuclear envelope membranes of isolated whole nuclei from human (panel (a)), rabbit (panel (f)), and rat (panel (k)) obtained after subtraction of the nucleoplasmic Syto-11 labeling ((b), (g), and (l) respectively) from the whole nucleus ((a), (f), and (k) respectively). Panels (d), (i), and (n) are the negative controls in the presence of blocking peptide and in the absence of the primary antibody. Panels (e), (j), and (o) are respectively the Syto-11 staining of nuclei of the experiments in panels (d), (i), and (i). The pseudocolour scale represents NHE-1 immunofluorescence level from 0 (black colour) to 255 (white colour). The white bar is 5 µm.

higher (13.02 ± 0.20, n = 5) than that of the cell membrane and the cytosol combined (11.45 ± 0.19, n = 5).

Discussion Our results, using immunofluorescence and 3-D confocal microscopy techniques, clearly showed for the first time the presence of NHE-1 at the level of nuclei isolated from both aortic and liver tissues. In addition, the fluorescent intensity labeling of the exchanger was more pronounced at the nuclear membranes. The level of NHE-1 in the whole nucleus seems to be similar in liver nuclei of rabbit and rat. However, the density of the exchanger in human liver nuclei is significantly higher than those found in rabbit and rat. This difference could be particularly due to a difference between species and to the fetal origin of human liver nuclei when compared with adult rabbit and rat livers. This latter possibility should be verified in the future. However, our results showed that the distribution and the level of fluorescence density of NHE-1 in isolated nuclei of aortic VSMCs of human were similar to those found in rat nuclei, but significantly different from those found in the aortic nuclei of the rabbit. These results using aortic and liver nuclei suggest that a difference or a similarity of nuclear membranes’ distri-

bution and levels of NHE-1 depends not only on the tissue origin of the nucleus, but also on the species origin of the tissue. Our results clearly showed that NHE-1 is also present at the nuclear envelope membranes of rat cardiomyocytes. The relative density of NHE-1 in ventricular cardiomyocytes seems to be higher at the whole nuclear level (including the nuclear envelope membranes) when compared with the density level of the sarcolemma membrane and the cytosol combined. The apparent low density of NHE-1 in the sarcolemma membrane and the cytosol combined, when compared with the whole nucleus, could be due to the very low density level of the exchanger at the cytosolic level. Due to the low density of the exchanger in the cytosol of heart cells, some middle sections showed almost an absence of exchanger labeling at the cytosolic level and clear labeling of the nuclear envelope membranes. Using these middle sections of the cells permitted us to estimate the relative density of NHE-1 at the sarcolemma level (without the cytosol) and the nuclear envelope membranes alone (after subtraction of the syto-11 nucleoplasmic labeling). The results suggested that the relative level of NHE-1 at the sarcolemma membrane of heart cells was significantly higher (p < 0.001) than that estimated for the nuclear envelope membranes. Whether the density of cell membrane NHE-1 is higher than that of the © 2004 NRC Canada

810 Fig. 3. Indirect immunofluorescence and 3-D reconstruction topview, look-through images of NHE-1 fluorescence intensity and distribution in cultured ventricular cardiomyocytes from 2-weekold rat (A). Panel (B) shows a pseudocolored middle cross- section of the cell in the lower part of panel A. The middle portion of the cell has been sectioned exposing the nucleus, with the remainder of the cell intact. As for isolated nuclei of aortic and liver tissues, labeling is associated with the plasma membrane and the nuclear envelope membranes, with weak labeling in the nucleoplasm and very weak labeling in the cytosol. The pseudocolored scale represents NHE-1 immunofluorescence level from 0 (black colour) to 255 (white colour). The white bar in (A) is 10 µm and in B is 5 µm.

Can. J. Physiol. Pharmacol. Vol. 82, 2004 Fig. 4. Immunofluorescence intensity levels of NHE-1 in isolated intact whole nuclei of aortic (A) and liver tissues (B) of human, rabbit, and rat as well as in the cytosol (including the plasma membrane) and nuclei (including the nuclear envelope membranes) of cultured rat ventricular cardiomyocytes (C). Values are means ± SEM. In panels (A) and (B), n is the numbers of different experiments from different aortas and each experiment represents the mean of 3 to 22 isolated nuclei that showed a clear staining with Syto-11. In panels (C), n represents the number of different experiments and each experiment is the mean of 3 to 5 cells that were stained with Syto-11 at the end of the experiment. PM is the plasma membrane, cyto is the cytoplasm, PNM is the perinuclear membranes, and NP is the nucleoplasm. *, p < 0.05 and ***, p < 0.001.

nuclear membranes and whether this difference could be cell type and (or) species dependent should be verified in the future. Our results also showed a fluorescent labeling of NHE1 at the cytosolic and nucleoplasmic levels of heart cells. This latter finding is expected since protein synthesis seems to also take place within the nucleus (Iborra et al. 2001; Bkaily et al. 2003b). Similar results were also reported for the presence in the cytosol and the nucleoplasm of receptors © 2004 NRC Canada

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such as ETB, AT1, and NPY Y1 (Bkaily et al. 2003a, 2003b; Jacques et al. 2003) as well as the Na+-Ca2+ exchanger (Xie et al. 2002). As for the plasma membrane (Karmazyn 2001; Karmazyn et al. 2003; Meng and Pierce 1991), the presence of NHE-1 at the level of the nuclear membranes may suggest that this exchanger may contribute not only to nuclear pH regulation, but may also contribute in the presence of nuclear membranes Na+-Ca2+ exchanger (Xie et al. 2002) to the modulation of nuclear Na+ and Ca2+ levels in normal and pathological conditions such as ischemia-reperfusion, hypertrophy, and heart failure (Karmazyn 2001; Karmazyn et al. 2003; Meng and Pierce 1991). Whether in these cardiac pathologies Na+ and Ca2+ overload takes place not only at the cytosolic levels (Karmazyn 2001; Karmazyn et al. 2003; Meng and Pierce 1991) but also at the nuclear levels and whether it is due to an increase in NHE-1 level and (or) activity should be verified in the future. More studies should be done on isolated nuclei from normal and diseased tissues, and more particularly heart muscle, to determine the functional aspect of nuclear membranes’ NHE-1 and its contribution to ischemiareperfusion, hypertrophy, and heart failure.

Acknowledgements This work was supported by a grant (MOP-37901) from the Canadian Institutes of Health Research. The authors acknowledge Mrs. Christiane Gauvin for her secretarial assistance.

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