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Spatial and Temporal Mg2+Signaling in Single Human Tracheal Gland Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
227, 743– 749 (1996)
1579
Spatial and Temporal Mg2/ Signaling in Single Human Tracheal Gland Cells Ste´phane Se´bille,* Jean-Marc Millot,* Michae¨l Maizie`res,† Maurice Arnaud,‡ Anne-Marie Delabroise,‡ Jacky Jacquot,† and Michel Manfait*,1 *Laboratoire de Spectroscopie Biomole´culaire, IFR 53, UFR de Pharmacie, 51, rue Cognacq Jay 51096 Reims, France; †INSERM U.314, IFR 53, Universite´ de Reims, 45, rue Cognacq Jay, 51092 Reims, France; and ‡Institut de l’Eau Perrier Vittel, 88804 Vittel, France Received August 2, 1996 The combined use of Mag-indo-1 probe and laser confocal UV-microspectrofluorometry allowed us to investigate the spatial and temporal dynamic changes of the Mg2/ variations in human tracheal gland (HTG) cells at the single cell level. Stimulation of HTG cells with either bradykinin, ouabain or extracellular high Mg2/ concentrations (up to 10 mM) induced increases in intracellular Mg2/ concentration [Mg 2/] i . From a cytosolic basal concentration of 0.8 { 0.3 mM in a medium free of Mg2/, an increase in extracellular Mg2/ concentration from 1 to 10 mM, increased cytosolic [Mg 2/] i from 1.4 { 0.6 to 1.8 { 0.8 mM after 10 min (põ0.05). We also demonstrated using line-scanned spectral images within single cells, that the [Mg2/ ]i is distributed uniformally in the nucleoplasm, but in contrast, showed marked local differences among different cytoplasmic regions, thus suggesting a functional heterogeneity in the intracellular Mg2/ stores involved. The influx pathway for Mg2/ in HTG cells was not inhibited by verapamil and appeared to be independent of [Ca2/] i . q 1996 Academic Press, Inc.
Human airway diseases including chronic bronchitis, asthma and cystic fibrosis are associated with excessive mucus production (1). Recently, we presented evidence (2,3) that intracellular free Ca2/ concentration ([Ca2/]i) oscillations in human tracheal gland (HTG) cells are at least one step in the cascade of events leading from secretagogue-receptor interaction to the induction of airway mucus secretion. In most instances, [Ca2/]i plays a central role in exocytosis process of numerous secretory cell types and marked differences in the sensitivity of [Ca2/]i and Ca2/ binding proteins have been reported (4,5). In parallel to the Ca2/-dependent signaling pathway linked to the exocytotic process, it has been recently proposed that cellular free magnesium, Mg2/, acts as a second messenger to mediate physiological responses (6). For example, Mg2/ is known to be closely linked to secretagogue-evoked Ca2/ mobilisation and secretory responses in pancreatic acinar cells (6). Other than its use as a co-substrate with ATP as Mg(ATP) and particularly in airway epithelial cells (7), potential roles of intracellular free Mg2/ concentration ([Mg2/]i) have not been studied in human airway epithelial cells, partly due to a lack of convenient methods for measuring dynamic changes of [Mg2/]i. To gain further insight into receptor-gate intracellular Mg2/ regulation, we used Mag-indo-1 fluorescence and laser scanning microspectrofluorometry to monitor dynamic changes of [Mg2/]i in single HTG cells attached to collagen-coated glass coverslip. Furthermore, spectral line scan images have been performed to better characterize the time resolution of [Mg2/]i dynamic changes throughout the whole cell. Utilizing this new quantitative confocal laser UV microspectrofluorometric method, we have addressed the following questions. 1) What is the basal [Mg2/]i in HTG cells ? 2) Are there any differences in the spatial (nucleoplasm versus cytosol) and temporal evolution of [Mg2/]i 1
To whom correspondence should be addressed. Fax: (33) 26 05 35 50. 743 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Reference spectra of intracellular Mag-indo-1. A : ratios of fluorescence intensities (I410/I 500) from cells loaded with Mag-indo-1 and permeabilized with 200 mM digitonin. At time 0, 50 mM extracellular Mg2/ (s) or 10 mM EDTA (l) was added. B : Cellular fluorescence spectra from Mg2/-permeabilized and Mag-indo-1 loaded cells in absence of Mg2/ (l), saturated with Mg2/ (s) and from unpermeabilized cells (n).
at the single cell level ? 3) What effects do high extracellular Mg2/ concentrations and exogenous pharmacological agonists of different classes (bradykinin, ouabain and bombesin) have on cellular dynamic changes [Mg2/]i ? MATERIALS AND METHODS Chemicals. Mag-indo-1 pentapotassium salt, Mag-indo-1/AM (prepared as stock solutions of 1mM dye), Indo-1/ AM, bradykinin, EDTA, ouabain, bombesin and verapamil were purchased from Sigma Chemical Co. (St Louis, MO). Cell culture. Primary cultures of human tracheal gland (HTG) cells were performed as previously described (3). Cells were subcultured onto 20 1 20 mm type 1 collagen coated glass coverslips in Dulbecco’s modified Eagle’s medium supplemented with 2% Ultroser G (Biosepra, Villeneuve la Garenne, France) in the presence of 2mM glutamine and antibiotics (penicillin 100 U/ml, streptomycin 100 mg/ml) under a humidified atmosphere of 5% CO2 95% air at 377c. The medium was changed after 2 days the cells were incubated until they became confluent and quiescent (6-7 days). In these culture conditions, cells exhibit characteristics of epithelial and serous secretory cell type (8) Mag-indo-1 loading. Quiescent cells on glass cover slips were washed twice with Dulbecco’s modified Eagle’s medium and incubated in medium containing 2 mM Mag-indo-1/AM for 45 min. at 377c. Microspectrofluorometer. Fluorescent emission spectra within an isolated living cell were recorded using a U.V. confocal microspectrofluorometer (DILOR, Lille, France). An optical microscope (Olympus BH2) was equipped with a water immersion objective lens (100X. N.A. 0.95, State Optical institute of St-Petersbourg, Russia). This objective lens has been specially developed for the total transmission of UV radiation down to 300 nm. The axial chromatic aberration was corrected, so that both excitation (330 nm) and emission (400-500 nm) voxels were superposed. The thickness of the optical section was controlled by varying the opening of a square pinhole from 50 to 1000 mm. The 351 nm laser line (Ar/, 2065A model, Spectra Physics) was focused with a measured power of 0.5 mW on the sample. The emission fluorescence 350-560 nm range was spectrally dispersed by a diffraction grating, and was detected with an optical multichannel analyser consisted of an air-cooled CCD detector (350-850nm range). [Mg 2/] i determination. [Mg2/] i was calculated according to the following equation (9): [Mg 2/] i Å b.Kd.(R min0R)/(R0Rmax) where R is the ratio of fluorescence intensity of the sample at 410 { 20 nm and 500 { 20 nm; Rmax and Rmin represent the ratios for Mag-indo-1 emissions in saturated and free of Mg 2/ conditions respectively; b is the emission intensity ratio of Mag-indo-1 at 500 nm in free and saturated Mg2/; Kd is the apparent dissociation constant of Mag-indo-1 for Mg2/. Cell to cell measurements of [Mg2/ ]i . Fields of 6-8 adjacent cells were vizualized on a video camera. The motorized 744
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TABLE 1 Mag-indo-1 Spectral Parameters Measured from Permeabilized HTG Cells (n Å 20) and Buffered Solutions
Solution: Nucleus:
Rmax
Rmin
b
Kd (mM)
0.8 { 0.01
0.18 { 0.01
2.4 { 0.05
2.8 { 0.05 (13)
0.9 { 0.05
0.24 { 0.05
#
#
0.95 { 0.05
0.24 { 0.05
#
#
Cell: Cytosol:
Note. Rmax and Rmin represent the ratios for mag-indo-1 emissions in saturated and free of Mg2/ respectively; b is the emission intensity ratio of mag-indo-1 at 500 nm in free and saturated conditions and Kd the dissociation constant of Mag-indo-1 for Mg2/.
stage (Ma¨rzha¨user, model MCL-2 with increments of 0.1 mM) coupled to a computer allowed to store the (x,y) positions of several chosen points (2,10). For measurements of [Mg2/ ]i dynamic changes, each point was in turn positioned for laser irradiation and measurement of the emission spectrum. Spectral scan images. Line scan images were obtained by the use of a microspectrofluorometer scanning system (DILOR, Lille, France) which provided line illumination innovation. Indeed, the line illumination system employed a system of two synchronized scanning mirrors that provided spectral accumulation from a lot of points simultaneously on a line (11). A horizontal line was virtually traced on a cell and spectra from each point of this line were recorded. Spectral line scan images with space as first dimension (x axis) and time as second dimension (y axis) were obtained. Spectral frame images were obtained in the same way with the use of a computer controlled motorized stage which displaced the sample in the y axis after each line acquisition. Spectra from cell edge were removed because of their low fluorescence intensity (below 2% of the maximum intensity).
RESULTS
In situ Reference Spectra of Mag-indo-1 For the [Mg2/]i measurements, reference spectra of Mag-indo-1 were established in single HTG cell from various intracellular microvolumes in saturated or free of Mg2/ conditions. These conditions were obtained after cell permeabilization using 200 mM digitonin and after
FIG. 2. Influence of [Ca2/ ]i on [Mg2/] i measurement. A : [Ca2/] i increase in HTG cells (nÅ5) after addition (time 0) of extracellular 5 mM Ca2/; B : [Mg 2/]i in HTG cells during the same period. 745
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FIG. 3. Intracellular Mg2/ distribution. A : a single HTG cell (bar Å 5 mM); B : integrated fluorescence intensity (400 – 500 nm) from this cell; C : [Mg2/ ]i within the cell.
the addition of either extracellular 50 mM Mg// or 10 mM EDTA (Fig. 1A). At the steadystate, fluorescence emission spectra were characterized by Rmax and Rmin values (ratios of emission intensity at 410 nm and 500 nm), for the saturated and free of Mg2/ conditions, respectively. Fig. 1B shows cellular fluorescence spectra of free Mg2/ Mag-indo-1 (l) and Mg2/ saturated Mag-indo-1 (s) in a permeabilized cell, as well as from an unpermeabilized cell (]) where both forms of the fluorescent dye were in equilibrium. Values of Rmin and Rmax were defined from different analyzed cells (nÅ20) and from buffered solutions (Table 1). According to these Rmax and Rmin values associated with the b.Kd constant defined from a buffered solution, the mean of basal [Mg2/]i of HTG cells was 0.8 { 0.3 mM (n Å 60). Influence of [Ca//]i Changes on Mag-indo-1 Emission As Mag-indo-1 was shown previously to bind Ca2/ (12), the putative interference by changes of [Ca2/]i in HTG cells was also investigated. To measure [Ca2/]i dynamic changes, HTG cells were loaded with 2 mM Indo-1/AM and then placed in 100 mM Hepes buffer pH Å 7.4 with low Ca2/ (0.5 mM). As shown in Fig. 2A, the addition of exogenous 5 mM Ca2/ induced
FIG. 4. A : Line scan image through a single HTG cell following the addition (time 0) of extracellular 8 mM Mg2/ B : time course changes of [Mg 2/]i observed in cytosol (n) and nucleus (l). Final [Mg2/] i in the cytosol and in the nucleus were significantly different (põ0.01). 746
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FIG. 5. Dynamic changes of [Mg2/ ]i in HTG cells after the addition (time 0) of 5 mM ouabain (nÅ5) (h); 5 mM bradykinin (nÅ5) (n); and 10 mM bombesin (nÅ5) (l).
a marked increase of [Ca2/]i from basal [Ca2/]i Å 50 { 15 nM to 180 { 35 nM. Moreover, no alteration in the measured basal [Mg2/]i was observed with these manoeuvres (Fig. 2B) reflecting a relative insensitivity of the Mag-indo-1 probe to Ca2/, in our experimental conditions. [Mg2/]i Spatial Distribution Fig. 3 shows an isolated HTG cell (Fig. 3A) and the loading intensity corresponding to integrated fluorescence in the 410-500 nm range (Fig. 3B). The Mag-indo-1 fluorescence emission was more important in the nucleus than in the cytosol. No significant heterogeneous fluorescence distribution was demonstrated in the cytosolic compartment. [Mg2/]i values obtained inside the whole cell are shown in the Fig. 3C. In the nucleoplasm, the mean of [Mg2/]i (0.9 { 0.3 mM, nÅ5) appeared homogenous and were slightly lower than [Mg2/]i values (1.2 { 0.5 mM, nÅ5) found in the cytosolic region. Most intriguingly, the confocal microspectrofluorometric method allowed to reveal a large spatial heterogeneity of cytosolic [Mg2/]i varying from one subcellular region (0.34 mM) to another opposite region (up to 3 mM) in the same HTG cell (Fig. 3C). [Mg2/]i Spatiotemporal Distribution after Agonist Stimulation Line scan images were performed to monitor dynamic changes in [Mg2/]i along a virtual line through a single HTG cell. Fig. 4A displays an example of a line scan image obtained after increasing the concentration of the extracellular Mg2/ from 0 to 8 mM Mg2/. As shown in Fig. 4B, the addition of external Mg2/ to the medium allowed the HTG cell to fill the [Mg2/]i stores up to 1.2 { 0.3 mM. Furthermore, differences between cytosol (1.37{ 0.2 mM) and nucleoplasm (1.13 { 0.15 mM) [Mg2/]i were observed over a period of 10 minutes. We next tested the ability of several physiological agonists to induce [Mg2/]i changes in HTG cells, by comparing the time-course changes of the [Mg2/]i induced by bradykinin, ouabain and bombesin. These agents are known to modify the ion transport processes in human airway epithelial cells (13). The addition of bradykinin (5 mM) and ouabain (5 mM) induced a significant increase in [Mg2/]i over a 12 min period (Fig. 5). By contrast, bombesin (10 mM) had no effect on the [Mg2/]i changes. 747
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FIG. 6. [Mg2/] i as a function of extracellular Mg2/ (h) (nÅ60). In 10 mM Mg2/ extracellular medium, [Mg2/] i was also determined in the presence of 25 mM of verapamil (VER,j) (nÅ60). (n, n : põ0.001) ; (*, * : põ0.001).
Investigation on a Mg2/ Influx Pathway To examine whether a Mg2/ influx pathway regulates the [Mg2/]i in HTG cells, the effect of three extracellular Mg2/ concentrations (0,1 and 10 mM) on [Mg2/]i was analyzed in single HTG cells. As shown in Fig. 6, an increase of extracellular Mg2/ concentration from 1 mM to 10 mM increased [Mg2/]i from 1.4 { 0.6 to 1.8 { 0.8 mM. As it can be observed, no linear correlation was demonstrated, suggesting thus, a highly regulated process of Mg2/ influx in HTG cells. In addition, we tested a common inorganic Ca2/-channel blocker, verapamil, to block the Mg2/ influx pathway. The addition of 25 mM verapamil did not modify the Mg2/ influx in HTG cells with high (10 mM) extracellular Mg2/ concentration (Fig. 6), leading credence to the notion that the Mg2/ influx pathway does not interfere with the Ca2/ influx pathway. DISCUSSION
Development of fluorescent probes for measuring [Mg2/]i and/or [Ca2/]i changes elicited by physiological agents has provided powerful tools to study dynamic changes in ion homeostasis during cell activation (14). The role of [Mg2/]i in the mechanisms of stimulus-response coupling of airway gland cells has been over shadowed by advancements in current knowledge about the second messenger role of [Ca2/]i and a comparable understanding of [Mg2/]i dynamic changes and Mg2/ homeostasis is currently lacking. At present, no data have been reported on [Mg2/]i changes in HTG cells in culture, evaluated by cell to cell studies to assess the heterogeneity of Mg2/ response at single cell level. For that, we developed a cell to cell analysis method which displayed the induced [Mg2/]i changes depending on each cell. Reference fluorescence emission spectra of Mag-indo-1 were obtained in both buffered solutions and single HTG cells (Fig. 1). Consistent with a previous study (12), the values of in situ constants for [Mg2/]i calibration agree with the Kd value calculated for Mg2/ (2.8 mM). The latter is optimal for monitoring physiological concentrations of free Mg2/ (0.5 to 10 mM). Fluorescent probes adapted for Mg2/ determination were suspected to respond to changes in [Ca2/]i in addition to changes in Mg2/ (15). In our experimental conditions, we did not observe any interference of [Ca2/]i changes in [Mg2/]i dynamic changes in HTG cells (Fig. 2). Thus, combined Mag-indo-1 and laser confocal UV microspectrofluorometry applied to individual cells offers several advantages over fluorometric techniques that employ cell suspensions, 748
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including temporal monitoring of single cell responses and analysis of spatial responses within single cells. In addition this method allows to answer to the questions that were evoked earlier. First, our results demonstrate that the mean of basal [Mg2/]i of HTG cells is 0.8 { 0.3 mM. Furthermore, the [Mg2/]i changes in single HTG cell were homogeneous in the nucleoplasm, but in contrast, showed a great spatial and temporal heterogeneity between two cytosolic regions of the cell (Fig. 3C). At last, [Mg2/]i was found to be dependent upon extracellular Mg2/ and moreover, the Mg2/ transport is sensitive to bradykinin and ouabain. Similar responses for the movement of Mg2/ have been reported in other cell types (15, 16). These complex responses are likely due to the release of Mg2/ from different intracellular stores. These stores may include mitochondria and negative charge ligands such as ATP, ADP, RNA and Mg2/ binding proteins (17). It has been reported that Mg2/ influx through plasma membrane showed similar characteristics to voltage-gate Ca2/ channels (18). Thus, depolarization solutions, which cancel transmembrane electrical potential and contain ouabain (Na/-ionophore) have been used to induce [Mg2/]i changes (19). In our study, the addition of ouabain in HTG cells induced [Mg2/]i changes (Fig. 5), which is in accordance with the previous statement. To summarize, experiments reported here made use of Mag-indo-1/AM loaded individual adherent HTG cells and a new method for the quantitation of dynamic changes of [Mg2/]i in single cells to demonstrate that a regulated process of [Mg2/]i levels exist in HTG cell. It remains to establish whether Mg2/ plays an important intracellular messenger role in the regulation of the mobilization of Ca2/ during the stimulus-secretion coupling process in HTG secretory cell. ACKNOWLEDGMENT The authors are thankful to G.D. Sockalingum for revision of the manuscript.
REFERENCES 1. Boat, T. F., Welsh, M. J., and Beaudet, A. L. (1969) in The Metabolic Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.), pp. 2649 – 2680, McGraw – Hill, New York. 2. Jacquot, J., Merten, M., Millot, J. M., Sebille, S., Me´nager, M., Figarella, C., and Manfait, M. (1995) Biochem. Biophys. Res. Commun. 212, 307– 316. 3. Jacquot, J., Maizie`res, M., Spilmont, C., Millot, J. M., Sebille, S., Merten, M., Kammouni, W., C., and Manfait, M. (1996) FEBS Lett. 386, 123– 127. 4. Davis, T. N. (1992) Cell 71, 557– 564. 5. Jessel, T. M., and Kandel, E. R. (1993) Cell 72, 1 –30. 6. Singh, J. and Wisdow, D. M. (1995) Mol. Cell. Biochem. 149, 175– 182. 7. Mason, S. J., Paradiso, A. M., and Boucher, R. C. (1991) Br. J. Pharmacol., 103, 1649 – 1656. 8. Jacquot, J., Spilmont, C., Burlet, H., Fuchey, Buisson, A. C., Tournier, J. M., Gaillard, D., and Puchelle, E. (1994) J Cell. Physiol. 161, 407– 418. 9. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440 –3450. 10. Millot, J. M., Pingret, L., Angiboust, J. F., Bonhomme, A., Pinon, J. M., and Manfait, M. (1995) Cell Calcium 17, 354– 366. 11. Sharonov, S., Chourpa, I., Morjani, H., Nabiev, I., and Manfait, M. (1994) Anal. Chim. Acta 290, 40– 47. 12. Morelle, B., Salmon, J. M., Vigo, V., and Viallet, P. (1993) Photochem. Photobiol. 58, 795– 802. 13. Boucher, R. C. (1994) Am. J. Respir. Crit. Care Med. 150, 271– 281. 14. Minamikawa, T. A., Takahashi, A., and Fujita, S. (1995) Cell Calcium 17, 165– 176. 15. Flatman, P. W. (1993) in Magnesium and Cell (Birch, N. J., Ed.), pp. 197– 216, Academic Press, London. 16. Gunther, T., Vormann, J., and Hollriegh, V. (1990) Biochem. Biophys. Acta 1023, 455 – 461. 17. Romani, A., Marfella, C., and Scarpa, A. (1992) Fed. Eur. Biochem. Soc. 296, 135– 140. 18. Dai, L. J., and Quamme, G. A. (1991) J. Clin. Invest. 88, 1255– 1264. 19. Zhang G. H., and Melvin J. E. (1995) FEBS Lett. 371, 52– 56.
749
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1579
Spatial and Temporal Mg2/ Signaling in Single Human Tracheal Gland Cells Ste´phane Se´bille,* Jean-Marc Millot,* Michae¨l Maizie`res,† Maurice Arnaud,‡ Anne-Marie Delabroise,‡ Jacky Jacquot,† and Michel Manfait*,1 *Laboratoire de Spectroscopie Biomole´culaire, IFR 53, UFR de Pharmacie, 51, rue Cognacq Jay 51096 Reims, France; †INSERM U.314, IFR 53, Universite´ de Reims, 45, rue Cognacq Jay, 51092 Reims, France; and ‡Institut de l’Eau Perrier Vittel, 88804 Vittel, France Received August 2, 1996 The combined use of Mag-indo-1 probe and laser confocal UV-microspectrofluorometry allowed us to investigate the spatial and temporal dynamic changes of the Mg2/ variations in human tracheal gland (HTG) cells at the single cell level. Stimulation of HTG cells with either bradykinin, ouabain or extracellular high Mg2/ concentrations (up to 10 mM) induced increases in intracellular Mg2/ concentration [Mg 2/] i . From a cytosolic basal concentration of 0.8 { 0.3 mM in a medium free of Mg2/, an increase in extracellular Mg2/ concentration from 1 to 10 mM, increased cytosolic [Mg 2/] i from 1.4 { 0.6 to 1.8 { 0.8 mM after 10 min (põ0.05). We also demonstrated using line-scanned spectral images within single cells, that the [Mg2/ ]i is distributed uniformally in the nucleoplasm, but in contrast, showed marked local differences among different cytoplasmic regions, thus suggesting a functional heterogeneity in the intracellular Mg2/ stores involved. The influx pathway for Mg2/ in HTG cells was not inhibited by verapamil and appeared to be independent of [Ca2/] i . q 1996 Academic Press, Inc.
Human airway diseases including chronic bronchitis, asthma and cystic fibrosis are associated with excessive mucus production (1). Recently, we presented evidence (2,3) that intracellular free Ca2/ concentration ([Ca2/]i) oscillations in human tracheal gland (HTG) cells are at least one step in the cascade of events leading from secretagogue-receptor interaction to the induction of airway mucus secretion. In most instances, [Ca2/]i plays a central role in exocytosis process of numerous secretory cell types and marked differences in the sensitivity of [Ca2/]i and Ca2/ binding proteins have been reported (4,5). In parallel to the Ca2/-dependent signaling pathway linked to the exocytotic process, it has been recently proposed that cellular free magnesium, Mg2/, acts as a second messenger to mediate physiological responses (6). For example, Mg2/ is known to be closely linked to secretagogue-evoked Ca2/ mobilisation and secretory responses in pancreatic acinar cells (6). Other than its use as a co-substrate with ATP as Mg(ATP) and particularly in airway epithelial cells (7), potential roles of intracellular free Mg2/ concentration ([Mg2/]i) have not been studied in human airway epithelial cells, partly due to a lack of convenient methods for measuring dynamic changes of [Mg2/]i. To gain further insight into receptor-gate intracellular Mg2/ regulation, we used Mag-indo-1 fluorescence and laser scanning microspectrofluorometry to monitor dynamic changes of [Mg2/]i in single HTG cells attached to collagen-coated glass coverslip. Furthermore, spectral line scan images have been performed to better characterize the time resolution of [Mg2/]i dynamic changes throughout the whole cell. Utilizing this new quantitative confocal laser UV microspectrofluorometric method, we have addressed the following questions. 1) What is the basal [Mg2/]i in HTG cells ? 2) Are there any differences in the spatial (nucleoplasm versus cytosol) and temporal evolution of [Mg2/]i 1
To whom correspondence should be addressed. Fax: (33) 26 05 35 50. 743 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Reference spectra of intracellular Mag-indo-1. A : ratios of fluorescence intensities (I410/I 500) from cells loaded with Mag-indo-1 and permeabilized with 200 mM digitonin. At time 0, 50 mM extracellular Mg2/ (s) or 10 mM EDTA (l) was added. B : Cellular fluorescence spectra from Mg2/-permeabilized and Mag-indo-1 loaded cells in absence of Mg2/ (l), saturated with Mg2/ (s) and from unpermeabilized cells (n).
at the single cell level ? 3) What effects do high extracellular Mg2/ concentrations and exogenous pharmacological agonists of different classes (bradykinin, ouabain and bombesin) have on cellular dynamic changes [Mg2/]i ? MATERIALS AND METHODS Chemicals. Mag-indo-1 pentapotassium salt, Mag-indo-1/AM (prepared as stock solutions of 1mM dye), Indo-1/ AM, bradykinin, EDTA, ouabain, bombesin and verapamil were purchased from Sigma Chemical Co. (St Louis, MO). Cell culture. Primary cultures of human tracheal gland (HTG) cells were performed as previously described (3). Cells were subcultured onto 20 1 20 mm type 1 collagen coated glass coverslips in Dulbecco’s modified Eagle’s medium supplemented with 2% Ultroser G (Biosepra, Villeneuve la Garenne, France) in the presence of 2mM glutamine and antibiotics (penicillin 100 U/ml, streptomycin 100 mg/ml) under a humidified atmosphere of 5% CO2 95% air at 377c. The medium was changed after 2 days the cells were incubated until they became confluent and quiescent (6-7 days). In these culture conditions, cells exhibit characteristics of epithelial and serous secretory cell type (8) Mag-indo-1 loading. Quiescent cells on glass cover slips were washed twice with Dulbecco’s modified Eagle’s medium and incubated in medium containing 2 mM Mag-indo-1/AM for 45 min. at 377c. Microspectrofluorometer. Fluorescent emission spectra within an isolated living cell were recorded using a U.V. confocal microspectrofluorometer (DILOR, Lille, France). An optical microscope (Olympus BH2) was equipped with a water immersion objective lens (100X. N.A. 0.95, State Optical institute of St-Petersbourg, Russia). This objective lens has been specially developed for the total transmission of UV radiation down to 300 nm. The axial chromatic aberration was corrected, so that both excitation (330 nm) and emission (400-500 nm) voxels were superposed. The thickness of the optical section was controlled by varying the opening of a square pinhole from 50 to 1000 mm. The 351 nm laser line (Ar/, 2065A model, Spectra Physics) was focused with a measured power of 0.5 mW on the sample. The emission fluorescence 350-560 nm range was spectrally dispersed by a diffraction grating, and was detected with an optical multichannel analyser consisted of an air-cooled CCD detector (350-850nm range). [Mg 2/] i determination. [Mg2/] i was calculated according to the following equation (9): [Mg 2/] i Å b.Kd.(R min0R)/(R0Rmax) where R is the ratio of fluorescence intensity of the sample at 410 { 20 nm and 500 { 20 nm; Rmax and Rmin represent the ratios for Mag-indo-1 emissions in saturated and free of Mg 2/ conditions respectively; b is the emission intensity ratio of Mag-indo-1 at 500 nm in free and saturated Mg2/; Kd is the apparent dissociation constant of Mag-indo-1 for Mg2/. Cell to cell measurements of [Mg2/ ]i . Fields of 6-8 adjacent cells were vizualized on a video camera. The motorized 744
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TABLE 1 Mag-indo-1 Spectral Parameters Measured from Permeabilized HTG Cells (n Å 20) and Buffered Solutions
Solution: Nucleus:
Rmax
Rmin
b
Kd (mM)
0.8 { 0.01
0.18 { 0.01
2.4 { 0.05
2.8 { 0.05 (13)
0.9 { 0.05
0.24 { 0.05
#
#
0.95 { 0.05
0.24 { 0.05
#
#
Cell: Cytosol:
Note. Rmax and Rmin represent the ratios for mag-indo-1 emissions in saturated and free of Mg2/ respectively; b is the emission intensity ratio of mag-indo-1 at 500 nm in free and saturated conditions and Kd the dissociation constant of Mag-indo-1 for Mg2/.
stage (Ma¨rzha¨user, model MCL-2 with increments of 0.1 mM) coupled to a computer allowed to store the (x,y) positions of several chosen points (2,10). For measurements of [Mg2/ ]i dynamic changes, each point was in turn positioned for laser irradiation and measurement of the emission spectrum. Spectral scan images. Line scan images were obtained by the use of a microspectrofluorometer scanning system (DILOR, Lille, France) which provided line illumination innovation. Indeed, the line illumination system employed a system of two synchronized scanning mirrors that provided spectral accumulation from a lot of points simultaneously on a line (11). A horizontal line was virtually traced on a cell and spectra from each point of this line were recorded. Spectral line scan images with space as first dimension (x axis) and time as second dimension (y axis) were obtained. Spectral frame images were obtained in the same way with the use of a computer controlled motorized stage which displaced the sample in the y axis after each line acquisition. Spectra from cell edge were removed because of their low fluorescence intensity (below 2% of the maximum intensity).
RESULTS
In situ Reference Spectra of Mag-indo-1 For the [Mg2/]i measurements, reference spectra of Mag-indo-1 were established in single HTG cell from various intracellular microvolumes in saturated or free of Mg2/ conditions. These conditions were obtained after cell permeabilization using 200 mM digitonin and after
FIG. 2. Influence of [Ca2/ ]i on [Mg2/] i measurement. A : [Ca2/] i increase in HTG cells (nÅ5) after addition (time 0) of extracellular 5 mM Ca2/; B : [Mg 2/]i in HTG cells during the same period. 745
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FIG. 3. Intracellular Mg2/ distribution. A : a single HTG cell (bar Å 5 mM); B : integrated fluorescence intensity (400 – 500 nm) from this cell; C : [Mg2/ ]i within the cell.
the addition of either extracellular 50 mM Mg// or 10 mM EDTA (Fig. 1A). At the steadystate, fluorescence emission spectra were characterized by Rmax and Rmin values (ratios of emission intensity at 410 nm and 500 nm), for the saturated and free of Mg2/ conditions, respectively. Fig. 1B shows cellular fluorescence spectra of free Mg2/ Mag-indo-1 (l) and Mg2/ saturated Mag-indo-1 (s) in a permeabilized cell, as well as from an unpermeabilized cell (]) where both forms of the fluorescent dye were in equilibrium. Values of Rmin and Rmax were defined from different analyzed cells (nÅ20) and from buffered solutions (Table 1). According to these Rmax and Rmin values associated with the b.Kd constant defined from a buffered solution, the mean of basal [Mg2/]i of HTG cells was 0.8 { 0.3 mM (n Å 60). Influence of [Ca//]i Changes on Mag-indo-1 Emission As Mag-indo-1 was shown previously to bind Ca2/ (12), the putative interference by changes of [Ca2/]i in HTG cells was also investigated. To measure [Ca2/]i dynamic changes, HTG cells were loaded with 2 mM Indo-1/AM and then placed in 100 mM Hepes buffer pH Å 7.4 with low Ca2/ (0.5 mM). As shown in Fig. 2A, the addition of exogenous 5 mM Ca2/ induced
FIG. 4. A : Line scan image through a single HTG cell following the addition (time 0) of extracellular 8 mM Mg2/ B : time course changes of [Mg 2/]i observed in cytosol (n) and nucleus (l). Final [Mg2/] i in the cytosol and in the nucleus were significantly different (põ0.01). 746
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FIG. 5. Dynamic changes of [Mg2/ ]i in HTG cells after the addition (time 0) of 5 mM ouabain (nÅ5) (h); 5 mM bradykinin (nÅ5) (n); and 10 mM bombesin (nÅ5) (l).
a marked increase of [Ca2/]i from basal [Ca2/]i Å 50 { 15 nM to 180 { 35 nM. Moreover, no alteration in the measured basal [Mg2/]i was observed with these manoeuvres (Fig. 2B) reflecting a relative insensitivity of the Mag-indo-1 probe to Ca2/, in our experimental conditions. [Mg2/]i Spatial Distribution Fig. 3 shows an isolated HTG cell (Fig. 3A) and the loading intensity corresponding to integrated fluorescence in the 410-500 nm range (Fig. 3B). The Mag-indo-1 fluorescence emission was more important in the nucleus than in the cytosol. No significant heterogeneous fluorescence distribution was demonstrated in the cytosolic compartment. [Mg2/]i values obtained inside the whole cell are shown in the Fig. 3C. In the nucleoplasm, the mean of [Mg2/]i (0.9 { 0.3 mM, nÅ5) appeared homogenous and were slightly lower than [Mg2/]i values (1.2 { 0.5 mM, nÅ5) found in the cytosolic region. Most intriguingly, the confocal microspectrofluorometric method allowed to reveal a large spatial heterogeneity of cytosolic [Mg2/]i varying from one subcellular region (0.34 mM) to another opposite region (up to 3 mM) in the same HTG cell (Fig. 3C). [Mg2/]i Spatiotemporal Distribution after Agonist Stimulation Line scan images were performed to monitor dynamic changes in [Mg2/]i along a virtual line through a single HTG cell. Fig. 4A displays an example of a line scan image obtained after increasing the concentration of the extracellular Mg2/ from 0 to 8 mM Mg2/. As shown in Fig. 4B, the addition of external Mg2/ to the medium allowed the HTG cell to fill the [Mg2/]i stores up to 1.2 { 0.3 mM. Furthermore, differences between cytosol (1.37{ 0.2 mM) and nucleoplasm (1.13 { 0.15 mM) [Mg2/]i were observed over a period of 10 minutes. We next tested the ability of several physiological agonists to induce [Mg2/]i changes in HTG cells, by comparing the time-course changes of the [Mg2/]i induced by bradykinin, ouabain and bombesin. These agents are known to modify the ion transport processes in human airway epithelial cells (13). The addition of bradykinin (5 mM) and ouabain (5 mM) induced a significant increase in [Mg2/]i over a 12 min period (Fig. 5). By contrast, bombesin (10 mM) had no effect on the [Mg2/]i changes. 747
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FIG. 6. [Mg2/] i as a function of extracellular Mg2/ (h) (nÅ60). In 10 mM Mg2/ extracellular medium, [Mg2/] i was also determined in the presence of 25 mM of verapamil (VER,j) (nÅ60). (n, n : põ0.001) ; (*, * : põ0.001).
Investigation on a Mg2/ Influx Pathway To examine whether a Mg2/ influx pathway regulates the [Mg2/]i in HTG cells, the effect of three extracellular Mg2/ concentrations (0,1 and 10 mM) on [Mg2/]i was analyzed in single HTG cells. As shown in Fig. 6, an increase of extracellular Mg2/ concentration from 1 mM to 10 mM increased [Mg2/]i from 1.4 { 0.6 to 1.8 { 0.8 mM. As it can be observed, no linear correlation was demonstrated, suggesting thus, a highly regulated process of Mg2/ influx in HTG cells. In addition, we tested a common inorganic Ca2/-channel blocker, verapamil, to block the Mg2/ influx pathway. The addition of 25 mM verapamil did not modify the Mg2/ influx in HTG cells with high (10 mM) extracellular Mg2/ concentration (Fig. 6), leading credence to the notion that the Mg2/ influx pathway does not interfere with the Ca2/ influx pathway. DISCUSSION
Development of fluorescent probes for measuring [Mg2/]i and/or [Ca2/]i changes elicited by physiological agents has provided powerful tools to study dynamic changes in ion homeostasis during cell activation (14). The role of [Mg2/]i in the mechanisms of stimulus-response coupling of airway gland cells has been over shadowed by advancements in current knowledge about the second messenger role of [Ca2/]i and a comparable understanding of [Mg2/]i dynamic changes and Mg2/ homeostasis is currently lacking. At present, no data have been reported on [Mg2/]i changes in HTG cells in culture, evaluated by cell to cell studies to assess the heterogeneity of Mg2/ response at single cell level. For that, we developed a cell to cell analysis method which displayed the induced [Mg2/]i changes depending on each cell. Reference fluorescence emission spectra of Mag-indo-1 were obtained in both buffered solutions and single HTG cells (Fig. 1). Consistent with a previous study (12), the values of in situ constants for [Mg2/]i calibration agree with the Kd value calculated for Mg2/ (2.8 mM). The latter is optimal for monitoring physiological concentrations of free Mg2/ (0.5 to 10 mM). Fluorescent probes adapted for Mg2/ determination were suspected to respond to changes in [Ca2/]i in addition to changes in Mg2/ (15). In our experimental conditions, we did not observe any interference of [Ca2/]i changes in [Mg2/]i dynamic changes in HTG cells (Fig. 2). Thus, combined Mag-indo-1 and laser confocal UV microspectrofluorometry applied to individual cells offers several advantages over fluorometric techniques that employ cell suspensions, 748
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including temporal monitoring of single cell responses and analysis of spatial responses within single cells. In addition this method allows to answer to the questions that were evoked earlier. First, our results demonstrate that the mean of basal [Mg2/]i of HTG cells is 0.8 { 0.3 mM. Furthermore, the [Mg2/]i changes in single HTG cell were homogeneous in the nucleoplasm, but in contrast, showed a great spatial and temporal heterogeneity between two cytosolic regions of the cell (Fig. 3C). At last, [Mg2/]i was found to be dependent upon extracellular Mg2/ and moreover, the Mg2/ transport is sensitive to bradykinin and ouabain. Similar responses for the movement of Mg2/ have been reported in other cell types (15, 16). These complex responses are likely due to the release of Mg2/ from different intracellular stores. These stores may include mitochondria and negative charge ligands such as ATP, ADP, RNA and Mg2/ binding proteins (17). It has been reported that Mg2/ influx through plasma membrane showed similar characteristics to voltage-gate Ca2/ channels (18). Thus, depolarization solutions, which cancel transmembrane electrical potential and contain ouabain (Na/-ionophore) have been used to induce [Mg2/]i changes (19). In our study, the addition of ouabain in HTG cells induced [Mg2/]i changes (Fig. 5), which is in accordance with the previous statement. To summarize, experiments reported here made use of Mag-indo-1/AM loaded individual adherent HTG cells and a new method for the quantitation of dynamic changes of [Mg2/]i in single cells to demonstrate that a regulated process of [Mg2/]i levels exist in HTG cell. It remains to establish whether Mg2/ plays an important intracellular messenger role in the regulation of the mobilization of Ca2/ during the stimulus-secretion coupling process in HTG secretory cell. ACKNOWLEDGMENT The authors are thankful to G.D. Sockalingum for revision of the manuscript.
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