| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ocki
* Institute of Biomedical and Biomolecular Sciences, School of Pharmacy and Biomedical Sciences, Portsmouth, UK;
Nencki Institute of Experimental Biology, Warsaw, Poland;
Friedrich-Baur Institute, Department of Neurology, Ludwig-Maximilians University, München, Germany; and
Department of Pharmacology, University of Cambridge, Cambridge, UK
2Correspondence: Institute of Biomedical and Biomolecular Sciences, St. Michael’s Building, White Swan Road, Portsmouth, PO1 2DT, UK. E-mail: darek.gorecki{at}port.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
ocki, K., Lien, C.-F., Jiang, T., Arkle, S., Brutkowski, W., Brown, J., Lochmuller, H., Simon, J., Barnard, E. A., Górecki, D. C. Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells.
Key Words: Duchenne muscular dystrophy • ATP • P2X receptor • myoblast • mdx mouse
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. While disruption of this arrangement (attributed with a role in strengthening the sarcolemma during contractile activity) contributes to DMD pathology, additional pathogenic mechanisms have been suggested. These include aberrant cell signaling, increased oxidative stress, recurrent muscle ischemia, and abnormal Ca2+ influx (1
, 2)
. DNA microarray analyses have revealed a number of newly implicated genes to be differentially regulated in dystrophic muscle (3
, 4)
. It now seems likely that the dystrophic phenotype is a consequence of the interaction of at least several of the aforementioned factors. Among those newly identified candidate genes were genes for some P2 subtypes of the ionotropic ATP receptors (3
4
5)
.
ATP has long been observed to potentiate excitation by acetylcholine (ACh) at the neuromuscular junction (6
, 7)
. Extracellular ATP is now known to act on both the P2Y and the P2X receptor classes: the P2Y receptors being G-protein-coupled receptors (GPCRs) while P2X receptors are transmitter-gated cation channels (8
, 9)
. In muscle cells, two functioning P2Y receptors—P2Y1 and P2Y2—have been identified in myotubes and at the neuromuscular junction (10
, 11,)
and expression of P2Y1, P2Y2, P2Y4, P2X2, P2X5, and P2X6 receptors has been observed at some stages of skeletal muscle development (10
11
12
13
14)
.
Dramatic changes have recently been documented in expression patterns of specific purinergic receptors in regenerating muscles of the dystrophic mdx mouse (12
, 15)
and in human DMD (15)
. It was therefore conceivable that the absence of dystrophin and the dystrophin-associated proteins in skeletal muscle could lead to abnormalities of P2X receptor functions and could contribute to dystrophic pathology in DMD. We have used immortalized myoblast cell lines, i.e., SC5 derived from mdx mouse and IMO, normal (dystrophin-positive) control myoblasts from the same parent mouse strain, as well as C2C12 normal mouse myoblasts. Using these, we sought to analyze potential changes in P2X receptor expression and function in dystrophic muscle. We found that the dystrophin-negative myoblasts are eminently more sensitive to ATP; possible origins of these differences are discussed.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell cultures
Mouse C2C12 myoblasts, the SC-5, immortalized mdx, and the IMO, immortalized, dystrophin-positive control cell lines were used. The latter two cell lines were derived from the "immorto mouse" or H2Kb-tsA58 line (16
, 17)
and cultured in Dulbecco’s modified Eagles’ medium (DMEM) supplemented with 20% fetal calf serum, 4 mM L-glutamine, 100 unit/mL penicillin, 100 µg/mL streptomycin, and 20 unit/mL murine 
-interferon (Invitrogen, San Diego, CA, USA) at 33°C. When cells reached 95% confluence, differentiation into myotubes was induced (where stated) by changing to differentiation medium containing 5% horse serum combined with the withdrawal of -interferon and incubation at 37°C. All media and reagents were purchased from Sigma (Poole, UK).
RT-PCR analysis
Total cellular RNA was extracted from mouse skeletal muscle and muscle cells using RNA Isolation System (Promega, Southampton, UK) and first strands prepared as described before (5
, 15)
. Cycling conditions were 94°C for 4 min, followed by 35 cycles of 94°C for 60 s, 58°C for 60 s, 72°C for 60 s with a final extension step of 72°C for 7 min. The primer sets for the P2X receptor transcript amplifications were essentially the same as published by Freedman et al. (18)
and Ekokoski et al. (19)
, except two new sets of P2X- specific primers (Table 1
).
|
PCR products were resolved by electrophoresis in 2% agarose gels and visualized by ethidium bromide staining. The authenticity of the PCR products was confirmed by sequencing.
To analyze expression of dystrophin and utrophin, specific sets of primers based on the sequences published by Koening et al. (20)
, Love et al. (21)
, and Lederfein et al. (22)
(see Table 1
for details) were used as described above.
Intracellular Ca2+ measurements in muscle cell lines in vitro
Muscle cells were cultured on glass coverslips in a 6-well plate (500,000 cells/well) for 48 h under conditions described above. Cells (70–80% confluent) were loaded with Fura 2 AM (Molecular Probes, Oregon) in culture medium for 15 min at 33°C in a 95% O2, 5% CO2 atmosphere. After two brief washes in the assay buffer (5 mM KCl, 1 mM MgCl2, 0.5 mM Na2HPO4, 25 mM HEPES, 130 mM NaCl, 1 mM pyruvate, 5 mM D-glucose, and 2 mM CaCl2; pH 7.4), the coverslips were mounted in a cuvette and maintained in the assay buffer at room temperature in RF5001PC spectrofluorimeter (Shimadzu). Fluorescence was recorded at 510 nm with excitation at 340/380 nm. At the end of each experiment the Fura 2 fluorescence was calibrated by addition of 4 µM ionomycin to determine maximal fluorescence. The effects of ATP were also measured as above but in the CaCl2-free buffer with addition of 0.05 mM EGTA. In some experiments the cells were treated first with 200 nM thapsigargin (Sigma) in calcium-containing medium to irreversibly mobilize intracellular Ca2+, then the responses to ATP or ADP were measured as described above. PPADS: the nonselective P2 receptor antagonist was used at 50 µM concentration. Coomassie Brilliant Blue G 250 (CBB) at 1 µM final concentration (at this concentration it predominantly blocks P2X7 receptor (23
, 24)
and 5 µM KN-62 (a noncompetitive P2X7 receptor antagonist) were applied 10 min prior to the addition of ATP. 10–50 µM zinc was also used. In this concentration range Zn2+ is a co-agonist of P2X2 through to P2X6 and antagonist of P2X7 and P2X1 receptors (8)
. Each experiment was repeated at least five times.
Antibodies
All antibodies were polyclonal. For details see Table 2
. In addition, antibodies to dystrophin (V-20, Santa Cruz Biotech, dilution 1:250) and utrophin (Mupa-2, epitope: amino acids 2543-2738, a gift from Dr. S. Brown, Imperial College, London, dilution 1:750) were used in Western blot. Actin antibody (Sigma) was used as a protein-loading control.
|
Western blot
Freshly collected mouse and rat tissues were crushed in liquid nitrogen and proteins extracted as described before (5)
. Cultures of myoblasts or myotubes were washed with ice-cold PBS (with 2 mM sodium-orthovanadate, 50 mM NaF) and the cells were gently scraped from flasks, centrifuged at 200 x g for 5 min at 15°C; the cell pellet was resuspended in boiling lysis buffer (10 mM Tris-HCl; pH 7.4, 1 mM sodium orthovanadate, 1% SDS), homogenized, then microwaved at a high setting for 5–10 s.
Protein samples were mixed with 1 volume of Laemmli sample buffer (Bio-Rad, Hemel Hempstead, UK) and 0.05% mercaptoethanol and heated to 95°C for 4 min, separated on 8–10% SDS-polyacrylamide gels, and electroblotted onto Hybond C membranes (Amersham Pharmacia, Little Chalfont, Bucks, UK). The blots were incubated with a blocking solution (5% nonfat milk powder in PBST: PBS/0.05% Tween 20) overnight at 4°C and probed with specific antibodies prepared in blocking solution for 2 h at room temperature. After three washes with PBST, membranes were incubated with horseradish peroxidase-conjugated secondary anti-rabbit or anti-goat IgG (1:10,000) at room temperature for 30 min. The specific signal was visualized using the ECL Plus kit (Amersham-Pharmacia Biotech, Piscataway, NJ, USA) after blot exposure to autoradiographic film or captured using the chemiluminescence imaging station Chemi-Genius 2 (Syngene, Cambridge). Exposure times within the linear range were used for comparisons. All experiments were repeated with similar results obtained throughout. As a negative control, antibodies were preincubated with their peptide antigens at RT for 1 h, centrifuged, and the supernatant was used as described above.
Immunocytochemistry
Cells were cultured on coverslips or in 8-well chamber slides (Labtech), rinsed, and fixed with ice-cold 4% paraformaldehyde in PBS for 10 min. Cold acetone and methanol fixation methods were also used for comparison. Slides were blocked in 5% normal goat serum (NGS) in PBS for 30 min at RT. After blocking, the cells were incubated with the appropriate P2X antibody (Table 2)
in PBS containing 5% NGS and 1.5% NaCl. In some experiments NaCl was omitted (low salt conditions). When primary antibodies against intracellular epitopes were used, 0.1% Triton X-100 was present throughout in the blocking and in the primary antibody labeling solutions to permeabilize the cells. Cells were washed in PBS containing 1.5% NaCl, and visualized with Alexa Fluor 488-labeled secondary antibody (Molecular Probes, Eugene, OR, USA). After washing sections were stained for 30 min at 37°C with ToPro (Molecular Probes) nuclear counterstain (diluted 1:1000 in PBS) and mounted in Vectashield (Vector Labs, Burlingame, CA, USA) mounting medium. In some experiments, instead of ToPro, Vectashield with DAPI was used for nuclear counterstaining. Confocal analysis was performed using an LSM 510 Meta microscope (Zeiss, Oberkochen, Germany). As a negative control, antibodies were preincubated with the respective peptide antigens, as described above, or the primary antibody was omitted. Other evidence for specificity of these antibodies is noted elsewhere (15)
.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A shows representative agarose gel analysis of PCR products obtained there. In the specific case of P2X6, amplification always resulted in two products from both cultured muscle cells and whole muscles. Sequence analysis of the smaller product showed that it arises from a mouse-specific alternative splicing of the P2X6 transcript. Details of this alternative splicing have been given elsewhere (15)
.
|
Because of the unexpected detection of the P2X7 transcript in both skeletal muscle and cultured muscle cells, two additional primer sets recognizing distinct parts of the P2X7 mRNA were used to confirm its presence there (Fig. 1B
). The P2X7 transcript was clearly present in both normal and dystrophic cells at the myoblast and myotube stages; no alternative splicing was found therein.
Differences in responses to ATP in normal and dystrophic myoblasts
Since we were able to show the potential expression of a number of P2X receptor subunits in myoblasts, we have tested for functional differences in responses to ATP between normal and dystrophic phenotypes.
Addition of 1 mM ATP to dystrophic myoblasts (SC5) in a buffer containing 2 mM CaCl2 resulted in an immediate and large increase in the intracellular Ca2+ level (Fig. 2
A). This effect was dose-dependent and higher ATP concentrations (up to 2 mM) caused further increase in Ca2+ levels (data not shown). In contrast, the same concentration of ATP caused a much smaller change in intracellular Ca2+ in the normal myoblasts (IMO), reaching only 
35% of that found in dystrophic cells (Fig. 2B
). Stimulation with ADP also produced increase in intracellular Ca2+ but this signal was significantly lower than that caused by equimolar ATP (Fig. 2A, B
). In Ca2+-free medium (Fig. 2C
) stimulation of IMO and SC5 cells with 1 mM ATP produced increased intracellular Ca2+ concentrations in both cell lines. The effect of ADP and the ATP-induced rise in intracellular Ca2+ levels in Ca2+-free medium indicates the involvement of Gq-linked P2Y receptors.
|
To confirm the involvement of P2Y receptors in that rise and to isolate also the P2X receptor component therein, thapsigargin was used to empty intracellular Ca2+ stores prior to addition of ATP (in the Ca2+-containing medium). When SC5 dystrophic muscle cells were preincubated with 200 nM thapsigargin, at the end of that Ca2+ discharge an addition of 1 mM ATP still resulted in further strong and extended increase in intracellular Ca2+, appearing after a delay of 1 min (Fig. 2D
). This effect was fully blocked by the nonselective P2 receptor antagonist PPADS (Fig. 3
F). In contrast, ATP had little effect on normal muscle cells after thapsigargin pretreatment (Fig. 2E
).
|
As the P2X7 transcript was found present in myoblasts we tested the effects of P2X7 agonists and antagonists on normal and dystrophic myoblasts pretreated with thapsigargin. 2'(3')-benzoylbenzoylATP (BzATP), a preferential agonist at rodent P2X7 and P2X5 receptors (25)
, very effectively stimulated Ca2+ flux into dystrophic (SC5) cells (Fig. 3A, B
) but produced no response in IMO cells (data not shown). This effect was observed only at high BzATP concentration (300 µM), with 100 µM ineffective. Specificity was shown by application of two P2X7 receptor antagonists, Coomassie Brilliant Blue G-250 (CBB) and KN-62. CBB, where tested in rodents is a potent antagonist at mouse or rat P2X7, less so at rat P2X2 and extremely weak at other rat P2X receptors (25
, 26)
. KN-62 is a noncompetitive selective P2X7 antagonist (27)
. CBB (1 µM) and KN-62 (5 µM) each completely blocked the Ca2+ accumulation evoked by ATP or BzATP in SC5 cells treated with thapsigargin (Fig. 3A-D
). Finally, 25 µM Zn2+ (an activator of P2X2-6 receptors but an antagonist at the P2X7 receptor) completely inhibited calcium influx (Fig. 3E
).
It has been reported that ADP in the presence of traces of ATP or after a brief stimulation with ATP becomes able to activate mouse P2X7 receptors in microglial cells (28)
. Although we found that ADP stimulation caused an immediate increase in intracellular Ca2+ (Fig. 2A, B
) this increase was abolished after pretreatment of the cells with thapsigargin (Fig. 2D, E
), suggesting that here a P2Y receptor rather than P2X7 is responsible for this ADP-induced effect.
The effects reported above are not attributable to the Ca2+-chelating effect of ATP since, even at 10 mM ATP, the thermodynamic activity of Ca2+ in the Krebs solution is only reduced to 0.5 mM and incubations of cells in Krebs solution containing that level of Ca2+ had no effect over the period involved when ATP was used (data not shown).
In summary, activation of P2Y receptors, causing release of Ca2+ from intracellular stores, appears to contribute to the observed elevation in intracellular Ca2+ levels in mouse myoblasts generally, while in the dystrophin-positive myoblasts it appears to be the leading mechanism producing the ATP-evoked effects. In contrast, the significantly higher Ca2+ rise in the dystrophic myoblasts was predominantly due to activation of P2X receptor(s) producing influx of extracellular Ca2+. The pharmacological profile of this receptor matches that of P2X7 as it is activated by BzATP and this activation is blocked by CBB, KN-62, and Zn2+.
Expression of the P2X receptor proteins
Western blots
As transcripts for all 7 P2X subunits were detected in dystrophic muscle cells and responses to ATP indicated the presence of functional P2X receptor, we used Western blot to further characterize its composition there. Two P2X7 subunit-specific antibodies, available from different commercial sources (Alamone and Santa Cruz Biotech) and recognizing different intracellular domains of the P2X7 protein, detected a single band of 68 kDa in dystrophic myoblasts (Fig. 4
). Since in our RT-PCR analyses we did not find any alternative splicing in the P2X7 transcripts that could otherwise be responsible for this size difference from the expected 80 kDa, the single band detected with these antibodies corresponds to the deglycosylated form of the P2X7 receptor protein in the path to internalization as described by Feng et al. (29)
. In contrast, in extracts of mouse (Fig. 4)
or rat brain (not shown), used as positive controls, both of those antibodies always detected a larger size band (80 kDa) This size corresponds to the fully glycosylated form of the P2X7 receptor subunit in the cell membrane (30)
. Preabsorption of each antibody with the respective peptide antigen completely abolished the signal (Fig. 4)
, while preabsorption with the peptide antigen specific to a different P2X subunit had no effect, confirming the specificity of these results. To exclude the possibility that P2X7 expression occurs in the immortalized cells only, we have analyzed primary normal and dystrophic muscle cells and found the same 68 kDa band, alone, in all of these (data not shown).
|
With antibodies specific to other P2X subunits, only P2X4 and very weak P2X5-specific bands were detected in protein extracts of SC5 cells. Western blot analysis of total cellular protein preparations with P2X4-specific antibody (Fig. 4)
revealed one main immunoreactive band of 40 kDa and two other bands of higher molecular mass (60–70 kDa), probably corresponding to alternatively glycosylated forms of this subunit since P2X4 has six potential glycosylation sites (9)
. This correlated well with our own data in C2C12 myoblasts (5)
and with the recent finding of this subunit in rat muscle (31)
. The P2X5 expression was very weak (not shown) and the presence of this subunit was in agreement with Ryten et al. data that demonstrated P2X5 in rat myoblasts (13
, 14)
.
Immunocytochemistry
Cultured dystrophic myoblasts were seen in confocal laser microscopy to be immunolabeled by both anti-P2X7 and P2X4 antibodies. In the undifferentiated cells this revealed a punctate distribution of fluorescence at the cell membranes and also strong fluorescence in the cytoplasm, the latter attributed to both synthesis and internalization of receptors in these dividing cells (Fig. 4)
. In differentiating SC5 cells (Fig. 4G
) there was an increase of P2X7 staining at the cell periphery, predominantly in areas of cell-to-cell contact. Since there has been recent controversy on the specificity of an Alomone anti-P2X7 antibody (32)
, as discussed below, we have used in our immunocytochemistry not only that Alomone antibody (raised to amino acids 567-595) but also another one to the extracellular domain, to cover different epitopes on the P2X7 protein, as detailed in Table 2
. The results as shown in Fig. 4
were obtained with both antibodies. The use of various antibody dilutions (ranging from 1:500 to 1:3000) and the presence of high or low salt concentrations in the antibody medium had no effect on the overall staining occurrence and pattern. Preabsorption of each antibody with its respective peptide antigen completely abolished the signal (Fig. 4)
while preabsorption with the peptide antigen for a nonspecific subunit had no effect (data not shown).
Analysis of dystrophin and utrophin expression in IMO and SC5 myoblasts
To confirm the dystrophic status of SC5 cells and to determine the expression levels of dystrophin and its autosomal homologue, utrophin, in IMO myoblasts, we have analyzed the expression profiles of the dystrophin transcripts in both cells. The mdx mice have a point mutation in exon 23 of the dystrophin gene (33)
, which results in premature termination of translation and rapid degradation of the full-length dystrophin transcript but does not interfere with production of the short dystrophin isoforms such as Dp71 (34)
. Indeed, using RT-PCR with specific primer sets we found that the full-length transcript was only expressed in IMO cells and is completely undetectable in SC5 myoblasts while the Dp71 mRNA was present in both cells (Fig. 5
A). However, our attempts to detect the full-length dystrophin protein in IMO cells using Western blot were unsuccessful (data not shown), confirming that levels of this protein in myoblasts are very low. In contrast, when we have analyzed the expression of the dystrophin homologue utrophin in the same samples, we found the transcript and the protein in both IMO and SC5 cells (Fig. 5B, C
).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 35)
and in rodents there is evidence for the presence and functioning of the Gq-linked P2Y1, and P2Y2 receptors in myotubes and adult muscle cells (10
, 11
, 36)
and for the presence of P2Y1 in myoblasts and P2Y1 and P2Y4 in myotubes (12)
. However, in the dystrophic myoblasts the most significant ATP response was that dependent on external Ca2+. This is seen from the Ca2+ rise in cells with emptied calcium stores; this ATP action is specifically a feature of the Ca2+-permeating cation channels of the P2X receptor family.
Elevated activity of P2X receptors in dystrophic muscle fibers could contribute to the dystrophic phenotype by influencing Ca2+ homeostasis and/or cell membrane permeability or by affecting muscle contractility (31)
. Therefore, we focused our attention on those receptors. Our RT-PCR analysis had indicated that muscle cells at the myoblast stage could contain mRNA for all of the seven known mammalian P2X receptor subunits (9)
. Three of these transcripts (P2X4, 5, and 6) had also been found by Ryten et al. (13)
in rat myoblasts, and the P2X2, P2X4, P2X5, and P2X6 proteins have been immunodetected at various stages of developing fibers in embryonic rat and mouse skeletal muscle sections (12
13
14
15
, 31)
. However, at the earlier undifferentiated myoblast stage (i.e., the stage when the functional analyses here were performed), we found only P2X4 and P2X7 proteins. The surprising detection of the P2X7 subunit agreed with the myoblast ATP responses just noted and with a recent study showing P2X7 receptors in C2C12 myoblasts before and during differentiation (37)
. We detected the P2X4 subunit earlier in C2C12 muscle cells (5)
. P2X5 was shown before in rat primary myoblasts (13
, 14)
. Here its levels were low and the functional significance of P2X5 subunit in myoblasts is unclear, since for both mouse (38)
and rat (39)
homomeric P2X5 receptors (unlike human P2X5 subunits), no functional P2X ion channels have been found at a significant level. However, Torres (40)
and Bo et al. (23)
. showed that such channels are readily formed by P2X5 only in heteromers, e.g., with P2X4, but never with the P2X7 found here.
The discrepancy between the RNA and protein data for the other subunits is not unusual and may stem from the extreme sensitivity of PCR amplification (able even to detect so-called illegitimate transcription), with protein levels being below the detection threshold for the immunolocalization methods used here. Some transcripts may not be translated efficiently or may not undergo correct post-translational modification in muscle cells.
For the nucleotide responses seen in dystrophic myoblasts, the P2X7 receptor is an interesting, albeit surprising, candidate. Our immunolocalization and Western blot data report the presence of this protein in both the cell membrane and the cytosol of such myoblasts. The smaller, 68 kDa protein detected in myoblasts corresponds to the form of P2X7 receptor found recently in cell lines and associated with receptor internalization and recycling (29)
. While the specificity of the anti-P2X7 antibodies used here has recently been questioned (32)
, it applied only to sites labeled in the brain: at a peripheral site examined, the specificity of those antibodies was confirmed and, for at least one antibody used here (directed to a C-terminal epitope), evidence for its specific immunocytochemical reaction with P2X7 has been given (41
, 42)
. There is also recent functional evidence for P2X7 at the brain sites (43)
. Moreover, we tested antibodies raised to three different epitopes on the P2X7 protein and two methods for their application: equivalent results were obtained in all cases, supporting its presence in dystrophic myoblasts.
Our pharmacological data support the notion that the P2X7 receptor contributes to the responses of dystrophic myoblasts to ATP. The Ca2+ influx was triggered more potently by BzATP, known as a much better P2X7 agonist than ATP (9)
, and was further blocked (Fig. 3)
by CBB or KN-62 or by Zn2+ (44)
. In combination, that pattern of pharmacology is fully specific for the P2X7 receptor (9
, 25
26
27
, 44
45
46)
, including that receptor in the mouse.
A high concentration (300 µM) of BzATP was required and still higher concentrations for ATP in this response. However, at the mouse P2X7 receptor, BzATP and ATP are known to be much less potent than at the rat P2X7 (45
, 46)
.
Still greater weakening is predicted here since the mdx mouse and its control line are of C57BL/10 origin (47)
, and a remarkable point mutation (P451L) in the P2X7 gene has been discovered in that strain (48)
. It greatly weakens the sensitivity for ATP and BzATP without changing their actions at the P2X7 receptor. We have confirmed by genotyping and DNA sequencing that the P2X7 locus in the IMO and SC5 cells used here indeed carries mutant leucine at position 451 (data not shown). BzATP and ATP levels required for full effect here agree with the mutant P2X7 sensitivities found by Adriouch et al. (48)
.
We conclude that the combined evidence is consistent with the presence of a functional mouse P2X7 receptor in dystrophic mouse myoblasts while P2X4 and P2X5 receptors may also be involved.
As normal myoblasts differentiate to form myotubes, other functional P2X subtypes become significantly expressed at their cell membranes, as shown for mouse, rat, and human cells by Cseri et al. (49)
and Collet et al. (50)
by their Ca2+ influx properties and rise in membrane conductance. These actions were evoked by ATP at high potency (EC50 20 µM) and weakly by BzATP, indicating that P2X receptors besides P2X7 are present. This agrees with P2X2, P2X4, and P2X5 appearing as rat myoblasts form myotubes (12
, 31)
. Unfortunately, our understanding of P2X receptors in skeletal muscle is incomplete, with some data indicating that P2X receptors disappear in normal adult muscle cells (12
, 15
, 49
, 50)
and others showing expression and functional significance of specific subunits in this tissue (31)
.
In muscle, as in other cells such as lymphocytes, ATP may have a dual role—low ATP concentrations having a signaling function leading to, for example, cell differentiation, while a sustained stimulation by high ATP concentrations may result in drastic changes in cell homeostasis. This highly regulated effect of ATP appears to be abnormal in dystrophic myoblasts.
The mechanism by which P2X receptors may be involved in the dystrophic pathology is an open question. Although an up-regulation of certain P2X subunits other than P2X7 has recently been found in regenerating dystrophic myofibers in situ (5
, 15
, 14)
, our results did not reveal any obvious up-regulation of P2X receptors at the dystrophic myoblast stage. One possible origin of altered P2X receptor function in dystrophic muscles is a decrease in the ecto-ATPase density at their surface caused by the absence of dystrophin. Ecto-nucleotidases (51)
are ubiquitous and are abundant on muscle cells, including myoblasts. During muscle injury, excessive exercise, or muscle disease, high levels of nucleotides are released into the extracellular space and abundant sarcolemmal ectoATPase activity forms a defense mechanism against overstimulation of ATP receptors. Dystrophin determines the organization of the dystrophin-associated protein (DAP) complex at the sarcolemma, and this is disrupted in the muscular dystrophies by mutations in either dystrophin or DAP genes (2
, 52)
. Betto et al. (53)
have provided evidence that a DAP complex member, 
-sarcoglycan (adhalin), is a Ca2+, Mg2+-ecto-ATPase. By its low affinity for ATP, it would be active only at high concentrations of ATP (54)
. Thus, elevated extracellular ATP levels could produce sustained activation of ATP receptors and hence Ca2+ overload. Skeletal muscles from dystrophic patients and mdx mice are indeed less able to regulate Ca2+ levels, and they exhibit elevated Ca2+ concentrations in the subsarcolemmal region (55
, 56)
. Increased Ca2+ levels in skeletal muscles can induce cell death by activating aberrant proteolysis or affecting metabolism in mitochondria (55
, 57)
. Opening of the P2X large cytolytic pore in sarcolemma would have other serious consequences for the cell homeostasis and could contribute to the "leaky membrane" seen in dystrophic muscle (58)
. Finally, abnormal P2X function in dystrophic muscle could contribute to damage by affecting muscle contractility (31)
.
Our data offer another unexpected insight into the pathogenesis of dystrophin deficiency. The dystrophic myoblasts clearly differ from normal cells in their responses to ATP. The IMO and SC5 mouse cells used in this study are highly homogeneous populations differing only in the presence or absence of a mutation in the dystrophin gene. However, until now the consensus was that dystrophin expression at the myoblast stage is very low or absent, and hence of no significance to early muscle development. We have clearly shown here this is not the case for their sensitivity to ATP. Intriguingly, a similar and unexplained relationship between P2X receptor function and the dystrophin complex was shown in an earlier study by Ferrari et al. (59)
. They found a striking difference in the response to extracellular ATP between lymphoblastoid cells cultured from DMD patients and those from normal subjects. Again, the DMD cells were eminently more sensitive to stimulation by ATP, and these changes were attributed to activation of a P2X7-type receptor. Normal lymphocytes contain a very small amount of dystrophin transcripts but no dystrophin protein. Overall, therefore, although the expression of dystrophin as well as of DAPs (including -sarcoglycan) increases with muscle cell differentiation (60
, 61)
, we propose that the small amounts of dystrophin (mRNA or protein) expressed at the normal myoblast stage are of functional significance.
The mdx dystrophin gene has a point mutation in exon 23 that results in a stop codon and produces premature termination of translation of the full-length dystrophin transcript (33)
and rapid transcript degradation via a nonsense-mediated mRNA decay mechanism (62)
. This mutation, however, does not interfere with production of the short dystrophin isoforms, of which the Dp71 (or G-dystrophin) isoform may be of particular importance since it is expressed in developing muscle (34)
. We have shown that mdx-derived SC5 cells lack full-length dystrophin but express the Dp71 transcript (Fig. 5)
; hence, the presence of Dp71 is insufficient to prevent the ATP-evoked increase in cytoplasmic Ca2+ concentrations. Equally, the presence of the autosomal homologue of dystrophin (utrophin or DRP-1) in dystrophic cells (Fig. 5)
was not sufficient to prevent the ATP-dependent effects in myoblast. Therefore, the role of low levels of full-length dystrophin in myoblasts and in nonmuscle tissues merits further investigation. Alternatively, regulatory effects of the full-length dystrophin transcript via recently discovered antisense mechanisms (63)
could be considered.
In view of the evidence presented above, we hypothesize that P2X receptors may be a significant contributor to pathogenic Ca2+ entry in dystrophic mouse muscle. If the same effect were established in human muscle cells, this would be a factor to be considered in the pathogenesis of DMD and LGMD muscular dystrophies. Since pretreatment of dystrophic muscle cells with drugs decreasing intracellular Ca2+ levels enhances survival of dystrophic myotubes (64)
, development of therapies aimed at decreasing the extracellular ATP levels or at inhibition of specific ATP receptors should be assessed for potential benefit in DMD and LGMD muscular dystrophies. Treatment decreasing extracellular ATP may be difficult in dystrophic muscle, which is permanently "leaking" ATP. Therefore, use of specific P2X antagonists may be a better strategy.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication July 26, 2005. Accepted for publication November 28, 2005.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-sarcoglycan (adhalin). J. Biol. Chem. 274,7907-7912This article has been cited by other articles:
|
|
 |
|
  Y. Iwata, Y. Katanosaka, T. Hisamitsu, and S. Wakabayashi Enhanced Na+/H+ Exchange Activity Contributes to the Pathogenesis of Muscular Dystrophy via Involvement of P2 Receptors Am. J. Pathol., November 1, 2007; 171(5): 1576 - 1587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Weigert, M. Dufer, P. Simon, E. Debre, H. Runge, K. Brodbeck, H. U. Haring, and E. D. Schleicher Upregulation of IL-6 mRNA by IL-6 in skeletal muscle cells: role of IL-6 mRNA stabilization and Ca2+-dependent mechanisms Am J Physiol Cell Physiol, September 1, 2007; 293(3): C1139 - C1147. [Abstract] [Full Text] [P |
