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2-Chloro-adenosine Induces a Glutamate-Dependent Calcium Response in C2C12 Myotubes
Biochemical and Biophysical Research Communications 277, 546 –551 (2000) doi:10.1006/bbrc.2000.3709, available online at http://www.idealibrary.com on
2-Chloro-adenosine Induces a Glutamate-Dependent Calcium Response in C2C12 Myotubes Claudio Frank,* ,1 Anna Maria Giammarioli,† Loredana Falzano,† Stefano Rufini,‡ Stefania Ceruti,§ Alessandra Camurri,§ Walter Malorni,† Maria Pia Abbracchio,§ and Carla Fiorentini† *Department of Pharmacology and †Department of Ultrastructures, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy; ‡Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00173 Rome, Italy; and §Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy
Received September 26, 2000
Adenosine and its derivatives may induce acute changes, i.e., injury and death, in muscle cells. In the present work, we evaluated the intracellular calcium concentration in C2C12 myogenic cells differentiated in vitro to form myotubes and exposed to a metabolically stable analogue of adenosine, 2-chloro-adenosine. The compound was able to significantly modify ionic homeostasis by sensitizing muscle cells to the excitatory amino acid glutamate. A single exposure to glutamate led to a marked increase in intracellular calcium level. This is the first demonstration that adenosine analogues can regulate muscle cell integrity and function via an indirect increase of intracellular calcium ions. © 2000 Academic Press Key Words: myogenic cells; adenosine; calcium; glutamate.
Adenosine is a neurotransmitter able to regulate the survival and death of many different cell types such as astrocytes, lymphocytes, thymocytes, myoblastic cells, and some immortalized cell lines (1– 4). We have previously demonstrated that the relatively metabolically stable adenosine analogue 2-chloro-adenosine (2CA) induces cell injury and apoptosis of both mononucleated resting myoblasts and polynucleated differentiated myotubes (2, 5). However, the mechanisms by which adenosine may lead to muscle cell damage and eventually apoptosis are only partially known. One of the main regulators of muscle cell fate is known to be represented by calcium ions (Ca 2⫹). Muscle cell contractility and functionality is in fact modulated by intracellular calcium concentration that also induces cell damage or death when abnormally increased (3). For instance, it has been reported that dystrophic muscle cells show an increase in both Ca 2⫹ influx and cytosolic To whom correspondence should be addressed. Fax: ⫹ 39 06 49387104. E-mail: [email protected]. 1
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Ca 2⫹ concentration (4) that may trigger the activation of a calcium-dependent protease, causing in turn the death of muscle cells (6, 7). With this in mind, we have explored the possibility that adenosine and its analogues could induce a change of the intracellular calcium level in muscle cells, either directly or following additional stimuli. To verify such a hypothesis we have used the myoblastic cell line C2C12, which can undergo differentiation in vitro by shifting the culture to medium containing low concentrations of mitogens. During this process, myoblasts withdraw from the cell cycle, express muscle-specific structural features and fuse into multinucleated myotubes. We have in particular evaluated if the previously utilized adenosine analogue 2CA (2) can act by modifying cytosolic calcium levels. Our results indicate that, in control myotubes, neither 2CA nor an excitatory aminoacid, such as glutamate (which is known to raise intracellular calcium levels in a variety of different cell types), can modify per se cytosolic calcium concentrations. By contrast, a 48 h exposure of myotubes to 2CA resulted in the appearance of sensitivity to glutamate, which induced a significant and transient increase of intracellular calcium concentration. MATERIALS AND METHODS Cell cultures and myotube differentiation. C2C12 cells were grown in Dulbecco’s modified Eagle medium supplemented with 20% fetal calf serum (GIBCO), 100 IU/ml penicillin, and 100 g/ml streptomycin in a humidified atmosphere of 5% CO 2 and 95% air at 37°C. Experiments were performed on cells with no more than 20 passages in culture. Myotube differentiation was induced at high confluence level by changing the medium to DMEM supplemented with 2% horse serum (GIBCO). Spindle-shaped cells in the first stages of differentiation to myotubes were observed after 24 h of incubation. Experiments were carried out with cells grown for 48 h in differentiating medium. The formation of myotubes was routinely checked by calculating the fusion index, which is represented by the number of cells counted at the inverted microscope/number of nuclei. Nonfused cultures have an index of 1 (100 cells/100 nuclei). A fusion index lower than 1 indicates that syncythia have been formed (e.g.,
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100 cells/500 nuclei ⫽ 0.2). Ideal fusion indexes tend towards “0”; in our experimental conditions, the fusion index was between 0.15 and 0.25. Drugs and cell treatments. Myotubes were exposed to 100 M 2-chloro-adenosine (2CA) for different time periods (8, 16, 24, and 48 h). Such a concentration was chosen on the basis of previous studies performed with this adenosine analogue (2). Evaluation of intracellular calcium was conducted in control and 2CA-treated cells as described below. In addition, an overnight exposure to the protein synthesis inhibitor cycloheximide (CHX) (Sigma) was performed by adding to myocells already treated with 2CA for 30 h, 5 g/ml CHX for 18 h before challenge with glutamate. Glutamate (Glu), trans-(1S,3R)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-(1S,3R)-ACPD), and ⫾-␣-methyl-4-carboxyphenylglycine (MCPG) were purchased from RBI (Research Biochemicals International); N-methyl-D-aspartate (NMDA), kainate, (S)-␣amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA), and D(⫺)-2-amino-5-phosphopentanoic acid (AP5), were purchased from Tocris Cookson Ltd. The substances were applied to the cells after 48 h of 2CA treatment. The concentrations used were: 1 mM Glu; 10 ⫺2, 10 ⫺1, and 1 mM for NMDA and AP5; 5 ⫻ 10 ⫺2, 5 ⫻ 10 ⫺1, and 1 mM for kainate and AMPA; 10 ⫺2, 10 ⫺1, and 1 mM for trans-ACPD and MCPG. To positively modulate the possibly present NMDA receptor, the application of NMDA was preceded by pretreatment with 100 M glycine. Intracellular calcium measurement. The bath solution (pH 7.35) contained (in mM): glucose 8, NaCl 125, KCl 1, CaCl 2 5, MgCl 2 1, Hepes 20. Glu (1 mM) was directly applied to the bath. Optical fluorimetric recordings with Fura 2AM were utilized to evaluate the intracellular calcium concentration ([Ca 2⫹] i). Cells were incubated for 50 min at room temperature with 5 M Fura-2 bath solution, that was then removed, replaced with extracellular solution, and the dishes were quickly placed on the microscope plate. Seven to 21 cells were analyzed at the same time. The Ca 2⫹ concentration was expressed as ratio between Fura-2 fluorescence at 340 and 380 nm excitation lights. A Leica DM IRB microscope and a Hamamatsu Argus 50 computerized analysis system were used to measure fluorescence. Statistical analysis was performed using Student’s t test. A P value lower than 0.01 was considered significant. Scanning electron microscopy. Controls and treated C2C12 cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 20 min. Following postfixation in 0.1% OsO 4 for 30 min, cells were dehydrated through graded ethanols, critical point-dried in CO 2 and gold-coated by sputtering. The samples were examined with a Cambridge 360 scanning electron microscope. Fluorescence microscopy. Controls and treated C2C12 cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS; pH 7.4) at room temperature for 10 min. After being washed in the same buffer, cells were permeabilized with 0.5% Triton X-100 (Sigma) for 10 min at room temperature. For F-actin detection, cells were stained with fluorescein-phalloidin (Sigma, working dilution 1:500) at 37°C for 30 min. The samples were analyzed with a Nikon Microphot fluorescence microscope.
RESULTS 2CA induces C2C12 myotubes to develop a calcium response to glutamate. To investigate the early damaging events induced by exposure of myocells to 2CA, we first evaluated the intracellular Ca 2⫹ homeostasis by means of fluorescence analyses. To this porpoise, we used C2C12 cells maintained in differentiation medium for 48 h until formation of myotubes and further
FIG. 1. Intracellular calcium measurement by fura-2AM in (a) untreated and (b) 2CA-treated myotubes. Addition of glutamate (1 mM) (arrows) induced calcium response only in 2CA-treated cells (b). Myotubes treated with 2CA for 0 to 12 h did not show sensitivity to Glu; a response appeared after 24 h in 16 (⫹/⫺4)% of treated cells (c). Treatment with 2CA for 48 h induced a Glu-sensitivity in 95 (⫹/⫺2)% of tested cells.
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FIG. 2. Scanning electron microscopy: control C2C12 cells showed the typical features of differentiated myotubes (a) which do not change after addition of glutamate (b). 2CA-treatment induced a dramatic disorganization of myotubes and the formation of numerous surface blebs (c). No further morphological changes were evident after challenging 2CA-treated cells with glutamate (d). Fluorescence microscopy: staining with fluorescein-phalloidin showed the well-organized stress fibers in untreated myotubes (e) which underwent breakdown after 2CA treatment (f ). Bar represents 10 m.
grown for 48 h in differentiation medium either in the presence or in the absence of 2CA. Although 2CA was unable per se to induce any alteration of intracellular calcium concentration at any time-point (data not shown), it sensitized myotubes to the excitatory aminoacid glutamate. The addition of glutamate (1 mM), in fact, although ineffective in modifying [Ca 2⫹] i in untreated myotubes (Fig. 1a), did evoke a powerful, fast and transient intracellular calcium increase in 2CA-treated myotubes occurring in 20 –30 min until a plateau was reached (Fig. 1b). Appearance of the calcium response to glutamate was indeed time-dependent, constantly occurring after 48 h of exposure to 2CA, occasionally after 24 h and never at shorter times of incubation with the drug (Fig. 1c). Regarding concentrations, we found that lower doses (10 M) of 2CA were not effective in inducing sensitivity to glutamate while higher concentrations could not be used because leading to a rapid, essentially necrotic, cell death (data
not shown). Furthermore, the appearance of the glutamate response could be fully prevented by the protein synthesis inhibitor cycloheximide, suggesting the need of protein synthesis to evoke the calcium response. 2CA provokes alterations of myotubes morphology and the breakdown of the actin cytoskeleton in C2C12 myotubes. As viewed by scanning electron microscopy, control myotubes showed the typical features of differentiated cells (Fig. 2a). Such morphology was unaffected by treatment with glutamate (Fig. 2b). Exposure to 100 M 2CA for 48 h profoundly altered the myotube morphology, cells losing the differentiated phenotype and showing the formation of numerous surface blebs (Fig. 2c). Such morphological features did not change after addition of glutamate (Fig. 2d). The morphological changes caused by 2CA were accompanied by a remarkable alteration of the actin cytoskeleton (Fig. 2f ), mainly consisting in the breakdown of
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FIG. 3. Application of Glu to myotubes treated with 2CA for 48 h induces an increase of [Ca 2⫹] i lasting for 20 –30 min (a). Application of the Glu ionotropic subtypes receptor agonists NMDA (b), AMPA (c), and kainate (d) and of the Glu metabotropic receptor agonist trans-ACPD, failed to elevate [Ca 2⫹] i (e). Pretreatment with the metabotropic-antagonist MCPG did not reduce Glu-sensitivity in 2CAtreated cells (f ).
the well-organized stress fibers that characterize untreated myotubes (Fig. 2e). Although to a lesser extent with respect to myoblastic undifferentiated cells (2), such alterations lead, prolonging the incubation time, to the detachment of cells from the substrate. In fact,
after 48 h of exposure to 2CA, it was evident a significant increase in the number of freely-floating cells in the culture medium (27% with respect to control cultures) as well as the number of detached cells showing signs of apoptosis (14% of floating cells).
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The glutamate-induced calcium response does not involve any of the known glutamate receptor subtypes. A series of experiments were therefore carried out to verify whether the induced calcium response to glutamate was mediated by a specific glutamate receptor subtype typically present, for instance, in neuronal cells (8, 9). We applied selective agonists and antagonists of these receptors to myotubes. NMDA (up to 1 mM, proceeded or not by 100 M glycine) failed to induce any change of intracellular calcium (Fig. 3b). In the same vein, application up to 1mM AMPA (Fig. 3c) or up to 1 mM kainate (Fig. 3d) did not provoke any increase in intracellular calcium. Finally, we evaluated the involvement of neuronal-like metabotropic receptors by either applying the metabotropic agonist t-ACPD (100 M) (Fig. 3e), or by pretreating cells with the antagonist MCPG (100 M) (Fig. 3f ). No effect was measured in the intracellular calcium concentration. DISCUSSION Exposure of various cell types (astrocytes, thymocytes, lymphocytes, and muscle cells) to excessive adenosine concentrations can trigger cell death (for a review see (10)). However, the mechanisms triggering apoptosis in muscle cells are still largely unexplored. On this basis, we have carried out experiments to investigate whether 2CA was able per se to induce muscle cell injury and death via modulation of intracellular calcium ionic homeostasis. In the present work we show that a significant [Ca 2⫹]i increase, followed by a partial recovery, occurred when 2CA-treated myotubes were exposed to glutamate. Interestingly, the intracellular calcium raise in muscle cells has been associated to changes in homocysteine homeostasis (11) and we have very recently reported the accumulation of S-adenosyl-homocysteine in muscle cells exposed to adenosine (5). Therefore, we can not rule out the possibility that 2CA-mediated cell death could also depend on the increase of both homocysteine and calcium. On the other hand, we cannot exclude a sort of “sensitization” to glutamate due to 2CA-mediated homocysteine accumulation. Moreover, the 2CA-induced alterations in myotubes were associated with the breakdown of the actin cytoskeleton whose network architecture is sensitive to calcium ion concentration (12). This can lead, as previously shown, to cell death by apoptosis (5) also suggesting the possibility of a 2CA-mediated alteration of muscle cell function. It is worth nothing that this transient calcium raise has peculiar features, being associated (or due) to muscle cell exposure to the excitatory aminoacid glutamate. Other features of 2CA-induced effects stand with the fact that i) the appearance of the glutamate response could be fully prevented by cycloheximide, suggesting the need of protein synthesis and ii) the glutamate-evoked calcium rise was not mediated
by either classic NMDA, AMPA, kainate, or metabotropic receptors. Although focused on C2C12 cultured cells differentiated in vitro, our experiments do have a few more general implications. On one side the intracellular calcium raise, when transitory, may provoke, in the shortcut, a cascade of intracellular events leading, in certain conditions (i.e., some metabolic or genetic diseases (13, 14)), to myotube functional alterations. In this case, however, the functionality of muscle cells is compatible with the survival. On the other side, when the calcium raise is irreversible, this leads in turn to that sequence of calcium-mediated effects that result in cell death by apoptosis. It has been hypothesized, for instance, that altered adenosine metabolism may be determinant for the degeneration associated to the Duchenne-type muscular dystrophy, where the levels of adenosine are markedly increased in both blood and muscles of patients (13, 14). Accordingly, muscular dystrophy associated degeneration has been reported to be also linked to a rise of calcium influx in muscle cells (4). In our opinion these data, although obtained by in vitro studies, may represent the first indication for a reappraisal of the role of adenosine in muscle cell patho-physiology. ACKNOWLEDGMENT The financial support of Comitato Telethon Fondazione Onlus (Grant 1050) is gratefully acknowledged.
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1. Cohen, A., Hirschhorn, R., Horowitz, S. D., Rubinstein, A., Polmar, S. H., Hong, R., and Martin, D. W. (1978) Deoxyadenosine triphosphate as a potentially toxic metabolite in adenosine deaminase deficiency. Proc. Natl. Acad. Sci. USA 75, 472– 476. 2. Rufini, S., Rainaldi, G., Abbracchio, M. P., Fiorentini, C., Capri, M., Franceschi, C., and Malorni, W. (1997) Actin cytoskeleton as a target for 2-chloro-adenosine: Evidence for induction of apoptosis in C2C12 myoblastic cells. Biochem. Biophys. Res. Commun. 238, 361–366. 3. Trump, B. F., and Berezesky, I. K. (1995) Calcium-mediated cell injury and cell death. FASEB J. 9, 219 –228. 4. Ruegg, U. T., and Gillis, J.-M. (1999) Calcium homeostasis in dystrophic muscle. TIPS 20, 351–352. 5. Ceruti, S., Giammarioli, A. M., Camurri, A., Falzano L., Rufini, S., Frank, C., Fiorentini, C., Malorni, W., and Abbracchio, M. P. (2000) Adenosine- and 2-chloro-adenosine-induced cytophatic effects on myoblastic cells and myotubes: involvement of different intracellular mechanisms. Neuromusc. Dis. 10, 436 – 446. 6. Turner, P. R., Westwood, T., Regen, C. M., and Steinhardt, R. A. (1988) Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice. Nature 335, 735–738. 7. Leijendekker, W. J., Passaquin, A. C., Metzinger, L., and Ruegg, U. T. (1996) Regulation of cytosolic calcium in skeletal muscle cells of the mdx mouse under conditions of stress. Br. J. Pharmacol. 118, 611– 616 8. Nicoletti, F., Bruno, V., Catania, M. V., Battaglia, G., Copani, A., Barbagallo, G., Cena, V., Sanchez-Prieto, J., Spano, P. F., and Pizzi, M. (1999) Group-I metabotropic glutamate receptors: Hy-
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potheses to explain their dual role in neurotoxicity and neuroprotection. Neuropharmacology 38, 1477–1484. 9. Nicoll, R. A., Malenka, R. C., and Kauer, J. A. (1990) Functional comparation of neurotransmitter receptor subtypes in mammalian central nervous system. Physiol. Rev. 70, 513–365. 10. Abbracchio, M. P., Ceruti, S., Brambilla, R., Franceschi, C., Malorni, W., Jacobson, K. A., von Lubitz, D. K., and Cattabeni, F. (1997) Modulation of apoptosis by adenosine in the central nervous system: A possible role for the A 3 receptor. Pathophysiological significance and therapeutic implications for neurodegenerative disorders. Ann. N. Y. Acad. Sci. 825, 11–22. 11. Mujumdar, V. S., Hayden, M. R., and Tyagi, S. C. (2000) Homo-
cyst(e)ine induces calcium second messenger in vascular smooth muscle cells. J. Cell. Physiol. 183, 28 –36. 12. Schliwa, M. (1981) Proteins associated with cytoplasmic actin. Cell 25, 587–590. 13. Castro-Gago, M., Lojo, S., Novo, I., del Rio, R., Pena, J., and Rodriguez-Segade, S. (1987) Effects of chronic allopurinol therapy on purine metabolism in Duchenne muscular dystrophy. Biochem. Biophys. Res. Commun. 147, 152–157. 14. Camina, F., Novo-Rodriguez, M. I., Rodriguez-Segade, S., and Castro-Gago, M. (1995) Purine and carnitine metabolism in muscle of patients with Duchenne muscular dystrophy. Clin. Chim. Acta 243, 151–164.
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2-Chloro-adenosine Induces a Glutamate-Dependent Calcium Response in C2C12 Myotubes Claudio Frank,* ,1 Anna Maria Giammarioli,† Loredana Falzano,† Stefano Rufini,‡ Stefania Ceruti,§ Alessandra Camurri,§ Walter Malorni,† Maria Pia Abbracchio,§ and Carla Fiorentini† *Department of Pharmacology and †Department of Ultrastructures, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy; ‡Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00173 Rome, Italy; and §Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy
Received September 26, 2000
Adenosine and its derivatives may induce acute changes, i.e., injury and death, in muscle cells. In the present work, we evaluated the intracellular calcium concentration in C2C12 myogenic cells differentiated in vitro to form myotubes and exposed to a metabolically stable analogue of adenosine, 2-chloro-adenosine. The compound was able to significantly modify ionic homeostasis by sensitizing muscle cells to the excitatory amino acid glutamate. A single exposure to glutamate led to a marked increase in intracellular calcium level. This is the first demonstration that adenosine analogues can regulate muscle cell integrity and function via an indirect increase of intracellular calcium ions. © 2000 Academic Press Key Words: myogenic cells; adenosine; calcium; glutamate.
Adenosine is a neurotransmitter able to regulate the survival and death of many different cell types such as astrocytes, lymphocytes, thymocytes, myoblastic cells, and some immortalized cell lines (1– 4). We have previously demonstrated that the relatively metabolically stable adenosine analogue 2-chloro-adenosine (2CA) induces cell injury and apoptosis of both mononucleated resting myoblasts and polynucleated differentiated myotubes (2, 5). However, the mechanisms by which adenosine may lead to muscle cell damage and eventually apoptosis are only partially known. One of the main regulators of muscle cell fate is known to be represented by calcium ions (Ca 2⫹). Muscle cell contractility and functionality is in fact modulated by intracellular calcium concentration that also induces cell damage or death when abnormally increased (3). For instance, it has been reported that dystrophic muscle cells show an increase in both Ca 2⫹ influx and cytosolic To whom correspondence should be addressed. Fax: ⫹ 39 06 49387104. E-mail: [email protected]. 1
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Ca 2⫹ concentration (4) that may trigger the activation of a calcium-dependent protease, causing in turn the death of muscle cells (6, 7). With this in mind, we have explored the possibility that adenosine and its analogues could induce a change of the intracellular calcium level in muscle cells, either directly or following additional stimuli. To verify such a hypothesis we have used the myoblastic cell line C2C12, which can undergo differentiation in vitro by shifting the culture to medium containing low concentrations of mitogens. During this process, myoblasts withdraw from the cell cycle, express muscle-specific structural features and fuse into multinucleated myotubes. We have in particular evaluated if the previously utilized adenosine analogue 2CA (2) can act by modifying cytosolic calcium levels. Our results indicate that, in control myotubes, neither 2CA nor an excitatory aminoacid, such as glutamate (which is known to raise intracellular calcium levels in a variety of different cell types), can modify per se cytosolic calcium concentrations. By contrast, a 48 h exposure of myotubes to 2CA resulted in the appearance of sensitivity to glutamate, which induced a significant and transient increase of intracellular calcium concentration. MATERIALS AND METHODS Cell cultures and myotube differentiation. C2C12 cells were grown in Dulbecco’s modified Eagle medium supplemented with 20% fetal calf serum (GIBCO), 100 IU/ml penicillin, and 100 g/ml streptomycin in a humidified atmosphere of 5% CO 2 and 95% air at 37°C. Experiments were performed on cells with no more than 20 passages in culture. Myotube differentiation was induced at high confluence level by changing the medium to DMEM supplemented with 2% horse serum (GIBCO). Spindle-shaped cells in the first stages of differentiation to myotubes were observed after 24 h of incubation. Experiments were carried out with cells grown for 48 h in differentiating medium. The formation of myotubes was routinely checked by calculating the fusion index, which is represented by the number of cells counted at the inverted microscope/number of nuclei. Nonfused cultures have an index of 1 (100 cells/100 nuclei). A fusion index lower than 1 indicates that syncythia have been formed (e.g.,
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100 cells/500 nuclei ⫽ 0.2). Ideal fusion indexes tend towards “0”; in our experimental conditions, the fusion index was between 0.15 and 0.25. Drugs and cell treatments. Myotubes were exposed to 100 M 2-chloro-adenosine (2CA) for different time periods (8, 16, 24, and 48 h). Such a concentration was chosen on the basis of previous studies performed with this adenosine analogue (2). Evaluation of intracellular calcium was conducted in control and 2CA-treated cells as described below. In addition, an overnight exposure to the protein synthesis inhibitor cycloheximide (CHX) (Sigma) was performed by adding to myocells already treated with 2CA for 30 h, 5 g/ml CHX for 18 h before challenge with glutamate. Glutamate (Glu), trans-(1S,3R)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-(1S,3R)-ACPD), and ⫾-␣-methyl-4-carboxyphenylglycine (MCPG) were purchased from RBI (Research Biochemicals International); N-methyl-D-aspartate (NMDA), kainate, (S)-␣amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA), and D(⫺)-2-amino-5-phosphopentanoic acid (AP5), were purchased from Tocris Cookson Ltd. The substances were applied to the cells after 48 h of 2CA treatment. The concentrations used were: 1 mM Glu; 10 ⫺2, 10 ⫺1, and 1 mM for NMDA and AP5; 5 ⫻ 10 ⫺2, 5 ⫻ 10 ⫺1, and 1 mM for kainate and AMPA; 10 ⫺2, 10 ⫺1, and 1 mM for trans-ACPD and MCPG. To positively modulate the possibly present NMDA receptor, the application of NMDA was preceded by pretreatment with 100 M glycine. Intracellular calcium measurement. The bath solution (pH 7.35) contained (in mM): glucose 8, NaCl 125, KCl 1, CaCl 2 5, MgCl 2 1, Hepes 20. Glu (1 mM) was directly applied to the bath. Optical fluorimetric recordings with Fura 2AM were utilized to evaluate the intracellular calcium concentration ([Ca 2⫹] i). Cells were incubated for 50 min at room temperature with 5 M Fura-2 bath solution, that was then removed, replaced with extracellular solution, and the dishes were quickly placed on the microscope plate. Seven to 21 cells were analyzed at the same time. The Ca 2⫹ concentration was expressed as ratio between Fura-2 fluorescence at 340 and 380 nm excitation lights. A Leica DM IRB microscope and a Hamamatsu Argus 50 computerized analysis system were used to measure fluorescence. Statistical analysis was performed using Student’s t test. A P value lower than 0.01 was considered significant. Scanning electron microscopy. Controls and treated C2C12 cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 20 min. Following postfixation in 0.1% OsO 4 for 30 min, cells were dehydrated through graded ethanols, critical point-dried in CO 2 and gold-coated by sputtering. The samples were examined with a Cambridge 360 scanning electron microscope. Fluorescence microscopy. Controls and treated C2C12 cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS; pH 7.4) at room temperature for 10 min. After being washed in the same buffer, cells were permeabilized with 0.5% Triton X-100 (Sigma) for 10 min at room temperature. For F-actin detection, cells were stained with fluorescein-phalloidin (Sigma, working dilution 1:500) at 37°C for 30 min. The samples were analyzed with a Nikon Microphot fluorescence microscope.
RESULTS 2CA induces C2C12 myotubes to develop a calcium response to glutamate. To investigate the early damaging events induced by exposure of myocells to 2CA, we first evaluated the intracellular Ca 2⫹ homeostasis by means of fluorescence analyses. To this porpoise, we used C2C12 cells maintained in differentiation medium for 48 h until formation of myotubes and further
FIG. 1. Intracellular calcium measurement by fura-2AM in (a) untreated and (b) 2CA-treated myotubes. Addition of glutamate (1 mM) (arrows) induced calcium response only in 2CA-treated cells (b). Myotubes treated with 2CA for 0 to 12 h did not show sensitivity to Glu; a response appeared after 24 h in 16 (⫹/⫺4)% of treated cells (c). Treatment with 2CA for 48 h induced a Glu-sensitivity in 95 (⫹/⫺2)% of tested cells.
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FIG. 2. Scanning electron microscopy: control C2C12 cells showed the typical features of differentiated myotubes (a) which do not change after addition of glutamate (b). 2CA-treatment induced a dramatic disorganization of myotubes and the formation of numerous surface blebs (c). No further morphological changes were evident after challenging 2CA-treated cells with glutamate (d). Fluorescence microscopy: staining with fluorescein-phalloidin showed the well-organized stress fibers in untreated myotubes (e) which underwent breakdown after 2CA treatment (f ). Bar represents 10 m.
grown for 48 h in differentiation medium either in the presence or in the absence of 2CA. Although 2CA was unable per se to induce any alteration of intracellular calcium concentration at any time-point (data not shown), it sensitized myotubes to the excitatory aminoacid glutamate. The addition of glutamate (1 mM), in fact, although ineffective in modifying [Ca 2⫹] i in untreated myotubes (Fig. 1a), did evoke a powerful, fast and transient intracellular calcium increase in 2CA-treated myotubes occurring in 20 –30 min until a plateau was reached (Fig. 1b). Appearance of the calcium response to glutamate was indeed time-dependent, constantly occurring after 48 h of exposure to 2CA, occasionally after 24 h and never at shorter times of incubation with the drug (Fig. 1c). Regarding concentrations, we found that lower doses (10 M) of 2CA were not effective in inducing sensitivity to glutamate while higher concentrations could not be used because leading to a rapid, essentially necrotic, cell death (data
not shown). Furthermore, the appearance of the glutamate response could be fully prevented by the protein synthesis inhibitor cycloheximide, suggesting the need of protein synthesis to evoke the calcium response. 2CA provokes alterations of myotubes morphology and the breakdown of the actin cytoskeleton in C2C12 myotubes. As viewed by scanning electron microscopy, control myotubes showed the typical features of differentiated cells (Fig. 2a). Such morphology was unaffected by treatment with glutamate (Fig. 2b). Exposure to 100 M 2CA for 48 h profoundly altered the myotube morphology, cells losing the differentiated phenotype and showing the formation of numerous surface blebs (Fig. 2c). Such morphological features did not change after addition of glutamate (Fig. 2d). The morphological changes caused by 2CA were accompanied by a remarkable alteration of the actin cytoskeleton (Fig. 2f ), mainly consisting in the breakdown of
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FIG. 3. Application of Glu to myotubes treated with 2CA for 48 h induces an increase of [Ca 2⫹] i lasting for 20 –30 min (a). Application of the Glu ionotropic subtypes receptor agonists NMDA (b), AMPA (c), and kainate (d) and of the Glu metabotropic receptor agonist trans-ACPD, failed to elevate [Ca 2⫹] i (e). Pretreatment with the metabotropic-antagonist MCPG did not reduce Glu-sensitivity in 2CAtreated cells (f ).
the well-organized stress fibers that characterize untreated myotubes (Fig. 2e). Although to a lesser extent with respect to myoblastic undifferentiated cells (2), such alterations lead, prolonging the incubation time, to the detachment of cells from the substrate. In fact,
after 48 h of exposure to 2CA, it was evident a significant increase in the number of freely-floating cells in the culture medium (27% with respect to control cultures) as well as the number of detached cells showing signs of apoptosis (14% of floating cells).
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The glutamate-induced calcium response does not involve any of the known glutamate receptor subtypes. A series of experiments were therefore carried out to verify whether the induced calcium response to glutamate was mediated by a specific glutamate receptor subtype typically present, for instance, in neuronal cells (8, 9). We applied selective agonists and antagonists of these receptors to myotubes. NMDA (up to 1 mM, proceeded or not by 100 M glycine) failed to induce any change of intracellular calcium (Fig. 3b). In the same vein, application up to 1mM AMPA (Fig. 3c) or up to 1 mM kainate (Fig. 3d) did not provoke any increase in intracellular calcium. Finally, we evaluated the involvement of neuronal-like metabotropic receptors by either applying the metabotropic agonist t-ACPD (100 M) (Fig. 3e), or by pretreating cells with the antagonist MCPG (100 M) (Fig. 3f ). No effect was measured in the intracellular calcium concentration. DISCUSSION Exposure of various cell types (astrocytes, thymocytes, lymphocytes, and muscle cells) to excessive adenosine concentrations can trigger cell death (for a review see (10)). However, the mechanisms triggering apoptosis in muscle cells are still largely unexplored. On this basis, we have carried out experiments to investigate whether 2CA was able per se to induce muscle cell injury and death via modulation of intracellular calcium ionic homeostasis. In the present work we show that a significant [Ca 2⫹]i increase, followed by a partial recovery, occurred when 2CA-treated myotubes were exposed to glutamate. Interestingly, the intracellular calcium raise in muscle cells has been associated to changes in homocysteine homeostasis (11) and we have very recently reported the accumulation of S-adenosyl-homocysteine in muscle cells exposed to adenosine (5). Therefore, we can not rule out the possibility that 2CA-mediated cell death could also depend on the increase of both homocysteine and calcium. On the other hand, we cannot exclude a sort of “sensitization” to glutamate due to 2CA-mediated homocysteine accumulation. Moreover, the 2CA-induced alterations in myotubes were associated with the breakdown of the actin cytoskeleton whose network architecture is sensitive to calcium ion concentration (12). This can lead, as previously shown, to cell death by apoptosis (5) also suggesting the possibility of a 2CA-mediated alteration of muscle cell function. It is worth nothing that this transient calcium raise has peculiar features, being associated (or due) to muscle cell exposure to the excitatory aminoacid glutamate. Other features of 2CA-induced effects stand with the fact that i) the appearance of the glutamate response could be fully prevented by cycloheximide, suggesting the need of protein synthesis and ii) the glutamate-evoked calcium rise was not mediated
by either classic NMDA, AMPA, kainate, or metabotropic receptors. Although focused on C2C12 cultured cells differentiated in vitro, our experiments do have a few more general implications. On one side the intracellular calcium raise, when transitory, may provoke, in the shortcut, a cascade of intracellular events leading, in certain conditions (i.e., some metabolic or genetic diseases (13, 14)), to myotube functional alterations. In this case, however, the functionality of muscle cells is compatible with the survival. On the other side, when the calcium raise is irreversible, this leads in turn to that sequence of calcium-mediated effects that result in cell death by apoptosis. It has been hypothesized, for instance, that altered adenosine metabolism may be determinant for the degeneration associated to the Duchenne-type muscular dystrophy, where the levels of adenosine are markedly increased in both blood and muscles of patients (13, 14). Accordingly, muscular dystrophy associated degeneration has been reported to be also linked to a rise of calcium influx in muscle cells (4). In our opinion these data, although obtained by in vitro studies, may represent the first indication for a reappraisal of the role of adenosine in muscle cell patho-physiology. ACKNOWLEDGMENT The financial support of Comitato Telethon Fondazione Onlus (Grant 1050) is gratefully acknowledged.
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