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Alteration of Free Calcium Levels and Acylphosphatase Muscular Isoenzyme in Cultured Dystrophic Skin Fibroblasts
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
230, 327–330 (1997)
RC965954
Alteration of Free Calcium Levels and Acylphosphatase Muscular Isoenzyme in Cultured Dystrophic Skin Fibroblasts D. Degl’Innocenti, A. Pieri, A. Berti, G. Liguri, C. Cecchi, and G. Ramponi1 Department of Biochemical Sciences, Universita` di Firenze, Viale Morgagni 50, 50134 Firenze, Italy
Received November 25, 1996
Levels of free intracellular calcium have been measured on two cell lines of cultured human fibroblasts carrying the genetic lesions occurring in Duchenne and Becker dystrophies. Both cell lines elicited a markedly higher content of the cation (98 nM and 57 nM, respectively) than control fibroblasts (35 nM). Differences toward controls were statistically significant (põ0.01). Dystrophic fibroblasts were also found to possess a significantly reduced amount by about 50% of muscular acylphosphatase isoenzyme as compared to normal cells. As acylphosphatase was demonstrated to be involved in the regulation of Ca2/-ATPase activity from different sources, a hypothesis was formulated that could explain the disruption of calcium homeostasis as an effect of the altered acylphosphatase activity. q 1997 Academic Press
the action mechanism of some calcium transport ATPases, notably those of erythrocyte membrane (3) and of heart sarcolemma (4). Acylphosphatase is a widespread cytosolic enzyme present in many mammalian tissues in two isoforms one of them named muscle type (MT), the other one common type (CT)(5). The two isoenzymes show similar structural and kinetics features but they appear to be differently involved in some physiological and pathological phenomena. Involvement of acylphosphatase in some differentiative processes has been demonstrated, particularly the MT isoform levels increase during myoblasts differentiation to myotubes (6). In this report , we investigated the relationship existing between abnormal calcium levels and acylphosphatase in human dystrophic fibroblast cultures. MATERIAL AND METHODS
During the past several years it has became clear that the perturbation of intracellular calcium homeostasis can play a determinant role in a variety of pathological cell conditions. Particularly the relationship between high calcium levels and various muscle diseases like dystrophy has been investigated by several authors. They demonstrated that [Ca2/]i is significantly elevated in mdx muscle fibers compared with normal fibers, both at rest and during stimulation (1). These abnormal ion levels in dystrophic muscle could result from a calcium influx caused by increased channel activity (2). The control of the Ca2/ levels in muscle fibers can be achieved by different systems, one of which is the Ca2/ transport associated to the activity of Ca2/ dependent ATPase. In our laboratory it has been demonstrated that the activity of this enzyme is modulated by acylphosphatase (EC 3.6.1.7.), that can hydrolyze the acylphosphate intermediates involved in 1
Corresponding author. Fax: /55 4222725.
In this work we used culture of dystrophic human skin fibroblasts (GM02298A and GM05162) and normal human skin fibroblasts The GM02298A (Coriell Institute) are fibroblasts derived from a 18 year male clinically affected Becker type dystrophy and the dystrophin gene shows no detectable deletion or duplication by PCR analysis. The GM05162 (Coriell Institute) are fibroblasts derived from 13 year male Duchenne dystrophy affected and the PCR analysis of dystrophin gene shows deletion starting at exon 46 or 47 though exon 48 or 49, exon 50 is not detected. All cell lines are grown in Dulbecco Modified Eagle’s Medium containing 10% foetal calf serum with the antibiotics penicillin and streptomycin in humidified atmosphere containing 6% CO2 . Cells were cultured in tissues culture dishes (Falcon) and seeded at a density of 11104 cell/cm2 until confluence. After removal the growth medium the confluent cells were washed with Puck’s solution for 2 min before detaching with 0,04% trypsine in Puck’s solution. Growth rate and cumulative population doubling at each passage were determined by counting cells in a Burker chamber. Cell viability was tested by Tripan-blue staining. Cells were resuspended in 1 ml of PBS and added to 1 ml of 1% Tripan-blue in PBS. After 10 minutes of incubation cells were screened for viability by visible- light microscopy. All cell lines were ranging between 9 and 20 cumulative population doubling. At different days of seeding, after removal of the growth medium, the cells were washed twice with ice-cold PBS, then collected in PBS
327
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE I
TABLE II
Muscular Acylphosphatase (MT) Levels in Cultured Skin Fibroblasts
Common Type Acylphosphatase (CT) Levels in Cultured Skin Fibroblasts
Controls GM02298A GM05162
Preconfluent
Confluent
2 days confluent
6.01 { 0.18 3.75 { 0.05 2.62 { 0.18
5.56 { 0.07 3.58 { 0.11 2.82 { 0.12
6.10 { 0.13 3.98 { 0.20 2.73 { 0.11
Controls GM02298A GM05162
Note. Results are the means { S.D. Data are expressed as ng of MT isoenzyme per mg of total protein. Differences between dystrophyc and control fibroblasts were highly significant (p õ 0.01) as assessed by Student’s t-test.
buffer containing 1mM PMSF, 10 mg/ml leupeptine and 10 mg/ml aprotinin. Cell suspension were sonicated and the lysates were centrifuged at 47C for 40 min at 29,0001g. Total protein contents were determined by BCA method (Pierce). Supernatant were assayed for acylphospatase isoenzymes by two different and specific non-competitive immunoenzymatic tests. Specific antibodies against the two acylphosphatase isoenzymes were obtained in rabbits using recombinant proteins (7,8), and purified by affinity chromatography (9). Free calcium determination was carried out using the fluorescent probe Fura2 (10). Subconfluent plates were accurately washed and gently trypsinized as above reported. After centrifugation cells were resuspended in DMEM supplemented with 10% FCS containing 10 mM Fura2-AM. In order to increase the availability of the probe, Fura2-AM stock solution (10 mM in dry DMSO) was precedently mixed 1:1 with 20% Pluronic F127. Incubation was performed at 37 7C for 60* after which the external dye was quickly removed by washing the cells with large excess of 25 7C 10 mM MOPS pH 7.0, containing 115 mM KCl, 20 mM NaCl, 1 mM EGTA, 1 mM MgCl2 . Cells were resuspended in 100 ml 10 mM MOPS pH 7.0, containing 115 mM KCl, 20 mM NaCl, 10 mM EGTA, 1 mM MgCl2 , counted and diluted in the same buffer to 1 1 106 cells/ml in a spectrofluorimetric termostated cuvette. Measurement was carried out at 257C and intracellular calcium concentration was derived from the equation
Preconfluent
Confluent
2 days confluent
4.28 { 0.05 3.88 { 0.04 4.42 { 0.03
4.57 { 0.11 4.24 { 0.06 4.72 { 0.03
4.70 { 0.08 4.46 { 0.09 4.31 { 0.04
Note. Results are the means { S.D. Data are expressed as ng of CT isoenzyme per mg of total protein.
of CT isoenzyme of acylphosphatase between dystrophic cells and control cells, as shown in Table II. The free intracellular calcium, [Ca2/]i , was studied in cultures of dystrophic skin fibroblasts GM02298A and GM05162 and of normal skin fibroblasts. Six different determinations on separate three days cultures of each cell line were carried out. Free intracellular calcium concentration was 56.8 nM and 97.6 nM in GM02298A and GM05162, respectively, as compared to 35.5 nM found in normal fibroblasts (Fig 1). Differences toward controls were statistically significant for both dystrophic cell lines. Fig 2 shows cell growth rates of the three cell lines under the same culture conditions as those for calcium and acylphosphatase assays. According to results found by other authors, fibroblasts with dystrophic character elicited slower growth rate as compared to normal con-
[Ca2/] Å KdQ(R0Rmin)/(Rmax0R) where R is the experimental value of the ratio F340/F380; Rmin and Rmax are respectively the R values at zero (10 mM Br-A23187 treated cells) and saturating (10 mM Br-A23187 and 50 mM calcium treated cells) calcium concentration; Q is the ratio Fzero/Fsaturating at 380 nm and Kd is the dissociation constant, 224 nM, according to Grynkiewicz et al. Statistical significance of the data were assessed by Student’s ttest.
RESULTS Cell content of acylphosphatase, CT and MT isoenzymes, was determined under different grade of confluence by immunoenzymatic assay. In Table I are reported results relative to the muscular acylphosphatase isoenzyme. These data evidentiate a markedly lower content of this enzyme in both dystrophic cell lines as compared to controls, the extent of decrease being more severe in Duchenne fibroblasts than in Becker fibroblasts. The significance of these differences was assessed be the Student t-test. On the contrary, no meaningful variation was found in the concentration
FIG. 1. Growth rate of skin human fibroblasts (l) and dystrophic human fibroblasts: GM02298 (n) and GM05162 (j). Cells seeded at 0.51104 / cm2 were grown and counted as described in Materials and Methods. Values are the means { S.D. of three different experiments. Cell lines were ranging between 15 and 20 cumulative population doubling.
328
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Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Free intracellular calcium levels in normal (A) and dystrophic human fibroblasts: cell lines GM02298 (B) and GM05162 (C). Results are the means { S.D. of six determinations in different experiments. * P õ 0.05 and ** P õ 0.01 in respect to controls.
trols. In this case too, the difference was wider for Duchenne fibroblasts than for Becker fibroblasts.
ried out a study on a cell type different from muscular fibres. As skin fibroblasts from human dystrophic subjects have been demonstrated to express dystrophin (14), whose defect is responsible for Duchenne and Becker muscular dystrophies, we used as a model two cell lines of human cultured fibroblasts carrying the genetic lesions occurring in Duchenne and Becker dystrophies. In vitro cultures of fibroblasts from patients with Duchenne muscular dystrophy exhibit some abnormal properties: decrease of intracellular adhesiveness (15), increased rate of cell-substratum detachment (16), abnormal spreading (17), altered growth kinetics (18), altered protein synthesis (19). From our results the evidence rises that dystrophic fibroblasts show a lower content, independent to rate growth, of the MT acylphosphatase isoenzyme and a higher level of free intracellular calcium. A direct causative relationship between calcium and acylphosphatase levels at present cannot be assessed. However, on the basis of previous results, demonstrating that acylphosphatase stimulates sarcoplasmic reticulum calcium pump (20), a mechanism can be suggested where the decrease of acylphosphatase activity leads to the higher free calcium concentration by the reduced stimulation of the endoplasmic reticulum calcium ATPase. On the other hand, in dystrophic cells the increase of free intracellular calcium could merely be an early event of pathology. Then the alteration of acylphosphatase content could be attributed to an increase of its degradation, possibly by the activation of intracellular calcium-proteases or by the downregulation of acylphosphatase expression. ACKNOWLEDGMENT
DISCUSSION
This work was supported by a grant from Telethon, Project 686.
A significant decrease of acylphosphatase activity in muscle biopsies from patients with Duchenne and other dystrophies was evidentiated by studies carried out in our Department (11,12). Previous experimental evidences also demonstrated an alteration of acylphosphatase levels in several physiological and pathological conditions where calcium homeostasis or transport is altered (6,11,13,). Several authors reported controversial results about the alterations of free calcium levels in muscle fibres from both mdx dystrophic mice and from subjects with Duchenne dystrophy (1,2). In these muscular models, several other histological and biochemical alterations have been evidentiated that could be indirectly responsible for impairment of calcium homeostasis. In order to clarify if the disruption of calcium homeostasis associated to muscular dystrophies and to confirm a possible functional relationship between acylphosphatase levels and calcium concentrations, we car-
REFERENCES 1. Turner, P. R., Westwood, T., Regen, C. M., and Steinhardt, R. A. (1988) Nature 335, 735–738. 2. Turner, P. R., Fong, P., Denetclaw, W. F., and Steinhardt, R. A. (1991) J. Cell Biol. 115, 1701–1712. 3. 735–738. 4. Nassi, P., Nediani, P., Liguri, G., Taddei, N., and Ramponi, G. (1991) J. Biol. Chem. 266, 10867–10871. 5. Nediani, C., Marchetti, E., Liguri, G., and Nassi, P. (1992) Biochem. Int. 26, 715. 6. Stefani, M., and Ramponi, G. (1995) Life Chemistry Reports, Vol. 12, 271–301. 7. Berti, A., Degl’Innocenti, D., Stefani, M., and Ramponi, G. (1992) Arch. Biochem. Biophys. 294, 261–265. 8. Modesti, A., Taddei, N., Bucciantini, M., Stefani, M., Colombini, B., Raugei, G., and Ramponi, G. (1995) Prot. Expr. Purif. 6, 799– 805. 9. Fiaschi, T., Raugei, G., Marzocchini, R., Chiarugi, P., Cirri, P., and Ramponi, G. (1995) FEBS Lett. 367, 145–148.
329
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6916$$$442
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AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10. Berti, A., Liguri, G., Stefani, M., Nassi, P., and Ramponi, G. (1982) Physiol. Chem. Phys. 14, 307. 11. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440–3450. 12. Nassi, P., Liguri, G., Landi, N., Berti, A., Stefani, M., Pavolini, B., and Ramponi, G. (1985) Biochem. Med. 34, 166–175. 13. Landi, N., Nassi, P., Liguri, G., Bobbi, S., Sbrilli, C., and Marconi, G. (1986) Clin. Chim. Acta 158, 245–251. 14. Liguri, G., Cecchi, C., Latorraca, S., Pieri, A., Sorbi, S., Degl’Innocenti, D., and Ramponi, G. (1996) Neurosci. Lett. 210, 153– 156. 15. Hugnot, J. P., Gilgenkrantz, H., Chafey, P., Lambert, M., Eveno,
16. 17. 18. 19. 20. 21.
E., Kaplan, J. C., and Kahn, A. (1993) Biochem. Biophys. Res. Comm. 192, 69–74. Jones, G. E., and Witkowski, J. A. (1993) Hum. Genet. 63, 232– 237. Kent, C. (1993) Proc. Natl. Acad. Sci. USA 80, 3087–3090. Pizzey, J., Witkowski, J. A., and Jones, G. (1987) J. Cell. Sci. 87, 163–169. Liechti-Gallati, S., Moser, H., Siegrist, H. P., and Herschkowitz, N. N. (1981) Pediatr. Res. 15, 1411–1414. Poche, H., and Schulze, H. (1985) J. Neurol. Sci. 70, 295–304. Nediani, C., Fiorillo, C., Marchetti, E., Pacini, A., Liguri, G., and Nassi, P. (1996) J. Biol. Chem. 271, 19066–19073.
330
AID
BBRC 5954
/
6916$$$442
12-24-96 01:16:52
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AP: BBRC
230, 327–330 (1997)
RC965954
Alteration of Free Calcium Levels and Acylphosphatase Muscular Isoenzyme in Cultured Dystrophic Skin Fibroblasts D. Degl’Innocenti, A. Pieri, A. Berti, G. Liguri, C. Cecchi, and G. Ramponi1 Department of Biochemical Sciences, Universita` di Firenze, Viale Morgagni 50, 50134 Firenze, Italy
Received November 25, 1996
Levels of free intracellular calcium have been measured on two cell lines of cultured human fibroblasts carrying the genetic lesions occurring in Duchenne and Becker dystrophies. Both cell lines elicited a markedly higher content of the cation (98 nM and 57 nM, respectively) than control fibroblasts (35 nM). Differences toward controls were statistically significant (põ0.01). Dystrophic fibroblasts were also found to possess a significantly reduced amount by about 50% of muscular acylphosphatase isoenzyme as compared to normal cells. As acylphosphatase was demonstrated to be involved in the regulation of Ca2/-ATPase activity from different sources, a hypothesis was formulated that could explain the disruption of calcium homeostasis as an effect of the altered acylphosphatase activity. q 1997 Academic Press
the action mechanism of some calcium transport ATPases, notably those of erythrocyte membrane (3) and of heart sarcolemma (4). Acylphosphatase is a widespread cytosolic enzyme present in many mammalian tissues in two isoforms one of them named muscle type (MT), the other one common type (CT)(5). The two isoenzymes show similar structural and kinetics features but they appear to be differently involved in some physiological and pathological phenomena. Involvement of acylphosphatase in some differentiative processes has been demonstrated, particularly the MT isoform levels increase during myoblasts differentiation to myotubes (6). In this report , we investigated the relationship existing between abnormal calcium levels and acylphosphatase in human dystrophic fibroblast cultures. MATERIAL AND METHODS
During the past several years it has became clear that the perturbation of intracellular calcium homeostasis can play a determinant role in a variety of pathological cell conditions. Particularly the relationship between high calcium levels and various muscle diseases like dystrophy has been investigated by several authors. They demonstrated that [Ca2/]i is significantly elevated in mdx muscle fibers compared with normal fibers, both at rest and during stimulation (1). These abnormal ion levels in dystrophic muscle could result from a calcium influx caused by increased channel activity (2). The control of the Ca2/ levels in muscle fibers can be achieved by different systems, one of which is the Ca2/ transport associated to the activity of Ca2/ dependent ATPase. In our laboratory it has been demonstrated that the activity of this enzyme is modulated by acylphosphatase (EC 3.6.1.7.), that can hydrolyze the acylphosphate intermediates involved in 1
Corresponding author. Fax: /55 4222725.
In this work we used culture of dystrophic human skin fibroblasts (GM02298A and GM05162) and normal human skin fibroblasts The GM02298A (Coriell Institute) are fibroblasts derived from a 18 year male clinically affected Becker type dystrophy and the dystrophin gene shows no detectable deletion or duplication by PCR analysis. The GM05162 (Coriell Institute) are fibroblasts derived from 13 year male Duchenne dystrophy affected and the PCR analysis of dystrophin gene shows deletion starting at exon 46 or 47 though exon 48 or 49, exon 50 is not detected. All cell lines are grown in Dulbecco Modified Eagle’s Medium containing 10% foetal calf serum with the antibiotics penicillin and streptomycin in humidified atmosphere containing 6% CO2 . Cells were cultured in tissues culture dishes (Falcon) and seeded at a density of 11104 cell/cm2 until confluence. After removal the growth medium the confluent cells were washed with Puck’s solution for 2 min before detaching with 0,04% trypsine in Puck’s solution. Growth rate and cumulative population doubling at each passage were determined by counting cells in a Burker chamber. Cell viability was tested by Tripan-blue staining. Cells were resuspended in 1 ml of PBS and added to 1 ml of 1% Tripan-blue in PBS. After 10 minutes of incubation cells were screened for viability by visible- light microscopy. All cell lines were ranging between 9 and 20 cumulative population doubling. At different days of seeding, after removal of the growth medium, the cells were washed twice with ice-cold PBS, then collected in PBS
327
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE I
TABLE II
Muscular Acylphosphatase (MT) Levels in Cultured Skin Fibroblasts
Common Type Acylphosphatase (CT) Levels in Cultured Skin Fibroblasts
Controls GM02298A GM05162
Preconfluent
Confluent
2 days confluent
6.01 { 0.18 3.75 { 0.05 2.62 { 0.18
5.56 { 0.07 3.58 { 0.11 2.82 { 0.12
6.10 { 0.13 3.98 { 0.20 2.73 { 0.11
Controls GM02298A GM05162
Note. Results are the means { S.D. Data are expressed as ng of MT isoenzyme per mg of total protein. Differences between dystrophyc and control fibroblasts were highly significant (p õ 0.01) as assessed by Student’s t-test.
buffer containing 1mM PMSF, 10 mg/ml leupeptine and 10 mg/ml aprotinin. Cell suspension were sonicated and the lysates were centrifuged at 47C for 40 min at 29,0001g. Total protein contents were determined by BCA method (Pierce). Supernatant were assayed for acylphospatase isoenzymes by two different and specific non-competitive immunoenzymatic tests. Specific antibodies against the two acylphosphatase isoenzymes were obtained in rabbits using recombinant proteins (7,8), and purified by affinity chromatography (9). Free calcium determination was carried out using the fluorescent probe Fura2 (10). Subconfluent plates were accurately washed and gently trypsinized as above reported. After centrifugation cells were resuspended in DMEM supplemented with 10% FCS containing 10 mM Fura2-AM. In order to increase the availability of the probe, Fura2-AM stock solution (10 mM in dry DMSO) was precedently mixed 1:1 with 20% Pluronic F127. Incubation was performed at 37 7C for 60* after which the external dye was quickly removed by washing the cells with large excess of 25 7C 10 mM MOPS pH 7.0, containing 115 mM KCl, 20 mM NaCl, 1 mM EGTA, 1 mM MgCl2 . Cells were resuspended in 100 ml 10 mM MOPS pH 7.0, containing 115 mM KCl, 20 mM NaCl, 10 mM EGTA, 1 mM MgCl2 , counted and diluted in the same buffer to 1 1 106 cells/ml in a spectrofluorimetric termostated cuvette. Measurement was carried out at 257C and intracellular calcium concentration was derived from the equation
Preconfluent
Confluent
2 days confluent
4.28 { 0.05 3.88 { 0.04 4.42 { 0.03
4.57 { 0.11 4.24 { 0.06 4.72 { 0.03
4.70 { 0.08 4.46 { 0.09 4.31 { 0.04
Note. Results are the means { S.D. Data are expressed as ng of CT isoenzyme per mg of total protein.
of CT isoenzyme of acylphosphatase between dystrophic cells and control cells, as shown in Table II. The free intracellular calcium, [Ca2/]i , was studied in cultures of dystrophic skin fibroblasts GM02298A and GM05162 and of normal skin fibroblasts. Six different determinations on separate three days cultures of each cell line were carried out. Free intracellular calcium concentration was 56.8 nM and 97.6 nM in GM02298A and GM05162, respectively, as compared to 35.5 nM found in normal fibroblasts (Fig 1). Differences toward controls were statistically significant for both dystrophic cell lines. Fig 2 shows cell growth rates of the three cell lines under the same culture conditions as those for calcium and acylphosphatase assays. According to results found by other authors, fibroblasts with dystrophic character elicited slower growth rate as compared to normal con-
[Ca2/] Å KdQ(R0Rmin)/(Rmax0R) where R is the experimental value of the ratio F340/F380; Rmin and Rmax are respectively the R values at zero (10 mM Br-A23187 treated cells) and saturating (10 mM Br-A23187 and 50 mM calcium treated cells) calcium concentration; Q is the ratio Fzero/Fsaturating at 380 nm and Kd is the dissociation constant, 224 nM, according to Grynkiewicz et al. Statistical significance of the data were assessed by Student’s ttest.
RESULTS Cell content of acylphosphatase, CT and MT isoenzymes, was determined under different grade of confluence by immunoenzymatic assay. In Table I are reported results relative to the muscular acylphosphatase isoenzyme. These data evidentiate a markedly lower content of this enzyme in both dystrophic cell lines as compared to controls, the extent of decrease being more severe in Duchenne fibroblasts than in Becker fibroblasts. The significance of these differences was assessed be the Student t-test. On the contrary, no meaningful variation was found in the concentration
FIG. 1. Growth rate of skin human fibroblasts (l) and dystrophic human fibroblasts: GM02298 (n) and GM05162 (j). Cells seeded at 0.51104 / cm2 were grown and counted as described in Materials and Methods. Values are the means { S.D. of three different experiments. Cell lines were ranging between 15 and 20 cumulative population doubling.
328
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12-24-96 01:16:52
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Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Free intracellular calcium levels in normal (A) and dystrophic human fibroblasts: cell lines GM02298 (B) and GM05162 (C). Results are the means { S.D. of six determinations in different experiments. * P õ 0.05 and ** P õ 0.01 in respect to controls.
trols. In this case too, the difference was wider for Duchenne fibroblasts than for Becker fibroblasts.
ried out a study on a cell type different from muscular fibres. As skin fibroblasts from human dystrophic subjects have been demonstrated to express dystrophin (14), whose defect is responsible for Duchenne and Becker muscular dystrophies, we used as a model two cell lines of human cultured fibroblasts carrying the genetic lesions occurring in Duchenne and Becker dystrophies. In vitro cultures of fibroblasts from patients with Duchenne muscular dystrophy exhibit some abnormal properties: decrease of intracellular adhesiveness (15), increased rate of cell-substratum detachment (16), abnormal spreading (17), altered growth kinetics (18), altered protein synthesis (19). From our results the evidence rises that dystrophic fibroblasts show a lower content, independent to rate growth, of the MT acylphosphatase isoenzyme and a higher level of free intracellular calcium. A direct causative relationship between calcium and acylphosphatase levels at present cannot be assessed. However, on the basis of previous results, demonstrating that acylphosphatase stimulates sarcoplasmic reticulum calcium pump (20), a mechanism can be suggested where the decrease of acylphosphatase activity leads to the higher free calcium concentration by the reduced stimulation of the endoplasmic reticulum calcium ATPase. On the other hand, in dystrophic cells the increase of free intracellular calcium could merely be an early event of pathology. Then the alteration of acylphosphatase content could be attributed to an increase of its degradation, possibly by the activation of intracellular calcium-proteases or by the downregulation of acylphosphatase expression. ACKNOWLEDGMENT
DISCUSSION
This work was supported by a grant from Telethon, Project 686.
A significant decrease of acylphosphatase activity in muscle biopsies from patients with Duchenne and other dystrophies was evidentiated by studies carried out in our Department (11,12). Previous experimental evidences also demonstrated an alteration of acylphosphatase levels in several physiological and pathological conditions where calcium homeostasis or transport is altered (6,11,13,). Several authors reported controversial results about the alterations of free calcium levels in muscle fibres from both mdx dystrophic mice and from subjects with Duchenne dystrophy (1,2). In these muscular models, several other histological and biochemical alterations have been evidentiated that could be indirectly responsible for impairment of calcium homeostasis. In order to clarify if the disruption of calcium homeostasis associated to muscular dystrophies and to confirm a possible functional relationship between acylphosphatase levels and calcium concentrations, we car-
REFERENCES 1. Turner, P. R., Westwood, T., Regen, C. M., and Steinhardt, R. A. (1988) Nature 335, 735–738. 2. Turner, P. R., Fong, P., Denetclaw, W. F., and Steinhardt, R. A. (1991) J. Cell Biol. 115, 1701–1712. 3. 735–738. 4. Nassi, P., Nediani, P., Liguri, G., Taddei, N., and Ramponi, G. (1991) J. Biol. Chem. 266, 10867–10871. 5. Nediani, C., Marchetti, E., Liguri, G., and Nassi, P. (1992) Biochem. Int. 26, 715. 6. Stefani, M., and Ramponi, G. (1995) Life Chemistry Reports, Vol. 12, 271–301. 7. Berti, A., Degl’Innocenti, D., Stefani, M., and Ramponi, G. (1992) Arch. Biochem. Biophys. 294, 261–265. 8. Modesti, A., Taddei, N., Bucciantini, M., Stefani, M., Colombini, B., Raugei, G., and Ramponi, G. (1995) Prot. Expr. Purif. 6, 799– 805. 9. Fiaschi, T., Raugei, G., Marzocchini, R., Chiarugi, P., Cirri, P., and Ramponi, G. (1995) FEBS Lett. 367, 145–148.
329
AID
BBRC 5954
/
6916$$$442
12-24-96 01:16:52
bbrcg
AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10. Berti, A., Liguri, G., Stefani, M., Nassi, P., and Ramponi, G. (1982) Physiol. Chem. Phys. 14, 307. 11. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440–3450. 12. Nassi, P., Liguri, G., Landi, N., Berti, A., Stefani, M., Pavolini, B., and Ramponi, G. (1985) Biochem. Med. 34, 166–175. 13. Landi, N., Nassi, P., Liguri, G., Bobbi, S., Sbrilli, C., and Marconi, G. (1986) Clin. Chim. Acta 158, 245–251. 14. Liguri, G., Cecchi, C., Latorraca, S., Pieri, A., Sorbi, S., Degl’Innocenti, D., and Ramponi, G. (1996) Neurosci. Lett. 210, 153– 156. 15. Hugnot, J. P., Gilgenkrantz, H., Chafey, P., Lambert, M., Eveno,
16. 17. 18. 19. 20. 21.
E., Kaplan, J. C., and Kahn, A. (1993) Biochem. Biophys. Res. Comm. 192, 69–74. Jones, G. E., and Witkowski, J. A. (1993) Hum. Genet. 63, 232– 237. Kent, C. (1993) Proc. Natl. Acad. Sci. USA 80, 3087–3090. Pizzey, J., Witkowski, J. A., and Jones, G. (1987) J. Cell. Sci. 87, 163–169. Liechti-Gallati, S., Moser, H., Siegrist, H. P., and Herschkowitz, N. N. (1981) Pediatr. Res. 15, 1411–1414. Poche, H., and Schulze, H. (1985) J. Neurol. Sci. 70, 295–304. Nediani, C., Fiorillo, C., Marchetti, E., Pacini, A., Liguri, G., and Nassi, P. (1996) J. Biol. Chem. 271, 19066–19073.
330
AID
BBRC 5954
/
6916$$$442
12-24-96 01:16:52
bbrcg
AP: BBRC