Pflügers Arch - Eur J Physiol (2002) 443:731–738 DOI 10.1007/s00424-001-0757-x
O R I G I N A L A RT I C L E
Julianna Cseri · Henrietta Szappanos Gyula Péter Szigeti · Zoltán Csernátony László Kovács · László Csernoch
A purinergic signal transduction pathway in mammalian skeletal muscle cells in culture Received: 2 August 2001 / Accepted: 31 October 2001 / Published online: 4 December 2001 © Springer-Verlag 2001
Abstract The effects of adenosine 5′-triphosphate (ATP) on human and mouse skeletal muscle fibres in primary culture were investigated. ATP-evoked changes in intracellular calcium concentration ([Ca2+]i) were measured and compared with those induced by agonists of the nicotinic acetylcholine (Ach)- and P2X purinoreceptors. While ATP was effective on both myoblasts and multinucleated myotubes in the micromolar range, Ach failed to induce any change in [Ca2+]i at early stages of development. In contrast, myofibres with peripheral nuclei showed little response to ATP but responded to Ach with a large change in [Ca2+]i. The responsiveness of the myotubes to Ach paralleled that to potassium. The removal of external calcium abolished the response to ATP. P2X receptor agonists mimicked the response to ATP with the order of potency being ATP>2′,3′O-(4-benzoyl)-benzoyl-ATP > β,γ-methylene-ATP > α,βmethylene-ATP. Under voltage-clamp conditions ATP induced an inward current that showed little inactivation. These results are consistent with the existence of P2X receptor-mediated signal transduction pathway in cultured mammalian skeletal muscle cells. Keywords Human · Skeletal muscle · Culture · ATP · Purinoreceptors · Development · Intracellular calcium
Introduction Considerable evidence indicates that adenosine 5′-triphosphate (ATP) is released from nerve terminals and acts as a co-transmitter in many tissues [7]. Extracellular J. Cseri · H. Szappanos · G.P. Szigeti · L. Kovács · L. Csernoch (✉) Department of Physiology, Medical and Health Science Centre, University of Debrecen, Debrecen, P.O.Box 22, 4012 Hungary e-mail:
[email protected] Tel.: +36-52-411717/5989, Fax: +36-52-432289 Z. Csernátony Department of Orthopaedic Surgery, Medical and Health Science Centre, University of Debrecen, Debrecen, P.O. Box 16, 4012 Hungary
ATP exerts its diverse effects by binding to membrane proteins termed P2 purinoreceptors [14]. P2 receptors have been classified into two families: a P2X family consisting of ligand-gated channels (at least seven subunits have been cloned [30, 33]) and a P2Y family consisting of G protein-coupled receptors. Individual P2X subunits with unknown stoichiometry form an essentially non-selective cation channel with an approximately twoto fourfold higher permeability for Ca2+ than for Na+ [16]. ATP activates cation channels in the membranes of fusion-competent myoblasts and myotubes obtained from chicken embryos [25]. This action of ATP was first believed to be mediated through binding to the nicotinic acetylcholine (Ach) receptor (nAchR). A similar conclusion has been drawn from measurements on cultured Xenopus laevis myotomal muscle cells [22] and on Xenopus oocytes expressing the nAchR from either BC3H1 cells (a skeletal muscle-type receptor) or from the Torpedo californica electric organ [15]. Work on embryonic chick skeletal muscle cells has, however, demonstrated that the ATP-induced inward currents represent an influx pathway that is independent of the nAchR [20] and is, most likely, mediated through the activation of P2X purinoreceptors [28, 36]. Recently, a novel gene associated with a P2X receptor, specifically expressed in human skeletal muscle, has been identified [37]. A similar gene and the encoded receptor have been found in the mouse with the receptor showing 83% homology to the human counterpart [29]. Examination of altered expressions of the human receptor suggests that it might play a significant role in the proliferation and/or differentiation of skeletal muscle cells. These findings indicate that a P2X receptor-mediated signal transduction mechanism might operate in developing skeletal muscle fibres in mammals and that it could play an important role in the regulation of cell differentiation and/or maturation. This and the accompanying paper [10] therefore test the hypothesis that ATP is involved in the regulation of intracellular calcium homeostasis in developing mamma-
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lian skeletal muscle via the activation of P2X purinoreceptors. We show, for the first time, that ATP induces an inward current and a transient rise in intracellular calcium concentration ([Ca2+]i) in cultured human and mouse skeletal muscle cells. The calcium transients were not mediated through the nAchR but were mimicked by P2X receptor agonists. These findings indicate the presence of a P2X receptor-mediated signalling pathway in developing mammalian skeletal muscle. This work has been presented to the Physiological Society [11].
Materials and methods Skeletal muscle cells in culture
ly as described for [Ca2+]i measurements. Cells were voltage clamped in the whole-cell mode of the patch-clamp technique using an amplifier (Axopatch 200A, Axon Instruments, Foster City, Calif., USA) as described earlier [32]. In brief, data was acquired using a TL1 interface in conjunction with an IBM-compatible computer and appropriate software (pClamp v. 6.0.4, Axon Instruments). Pipettes were pulled from borosilicate glass capillaries (BioLogic, Germany) and had resistances of 2–3 MΩ when filled with an artificial internal solution, containing (in mM): 110 Kaspartate, 20 KCl, 2 MgCl2, 5 EGTA, 5 HEPES and 2 MgATP (pH 7.3 using KOH). The passive electrical parameters of the cells were determined with 40-ms, 5-mV pulses. The holding potential was –80 mV. The linear capacitance of the cells was 150–200 pF. The effect of externally applied ATP was followed by continuously recording the holding current necessary to maintain the –80 mV holding potential. Experimental procedure and chemicals
Muscle tissues were obtained either from healthy human subjects undergoing orthopaedic surgery or from young mice in accordance with the guidelines and an approved protocol of the Ethics Committee of the University of Debrecen. The procedure for obtaining satellite cells from the samples and growing myotubes from the satellite cells is described elsewhere [5, 32]. In brief, the muscle biopsy was dissociated at 37 °C using collagenase (Type II, Sigma, St. Louis, Mo., USA) and trypsin (Difco, Detroit, Mich., USA) in a calcium/magnesium-free phosphate buffer. After filtration and centrifugation, the pellet was resuspended in Ham’s F-12 growth medium (Sigma) supplemented with 5% FCS, 5% horse serum (HS), 2.5 mg/ml glucose, 0.3 mg/ml glutamate, 1.2 mg/ml NaHCO3, 50 U/ml penicillin, 50 µg/ml streptomycin and 1.25 µg/ml fungizone (Biogal, Debrecen, Hungary). The cells were seeded onto sterile cover-slips (32 mm diameter, 0.07 mm thick; Biophysical Technologies, Sparks, Md., USA) and kept in a 5% CO2 atmosphere at 37 °C. After 3 days in culture the medium was changed to DMEM (Sigma) supplemented with 2% FCS and 2% HS to facilitate myoblast fusion and differentiation.
Statistical analysis
Assay of [Ca2+]i
ATP-induced changes in [Ca2+]i
Experiments were carried out on 7- to 14-day-old cultures for human and 5- to 8-day-old cultures for mice, carefully registering the degree of fusion for each myotube by counting the number of nuclei in the given cell. To introduce the calcium-sensitive probe into the myoplasmic space cells were incubated with 5 µM fura-2 AM (Molecular Probes, Eugene, Ore., USA) in the presence of 150 nM neostigmine (Pharmamagist, Budapest, Hungary) for 1 h at 37 °C. Before each measurement the myotubes were kept at room temperature (22–24 °C) in normal Tyrode’s solution (in mM): 137 NaCl, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2, 11.8 HEPES-NaOH, 1 g/l glucose, pH 7.4 for 30 min to allow homogeneous distribution of the dye. The cover-slips with the fura-2 AM-loaded cells were then placed on the stage of an inverted fluorescence microscope (Diaphot, Nikon, Japan). Excitation wavelength was altered between 340 and 380 nm by a dual-wavelength monochromator and an on-line connected microcomputer (Deltascan, Photon Technology International, New Brunswick, N.J., USA) while the emission was monitored at 510 nm using a photomultiplier at an acquisition rate of 10 Hz per ratio. [Ca2+]i levels were calculated according to [18] from the ratio of the fluorescence intensities measured at excitation wavelengths of 340 and 380 nm as described earlier [3] using in vivo calibration data. Voltage-clamp measurements For electrophysiological experiments the myotubes were kept in normal Tyrode’s solution at room temperature (22–24 °C), similar-
Agonists were applied through a fast perfusion system that allowed rapid (delay approximately 2.5 s) and local application of the compound onto the cell investigated [3]. All experiments for measuring [Ca2+]i were performed in either normal Tyrode’s solution or in a calcium-free Tyrode’s solution of the same composition as the normal Tyrode’s except for 5 mM EGTA and no added CaCl2. ATP and other chemicals were from Sigma.
Data are presented as means±SEM or as best fit value±SEM in non-linear curve fits. Where appropriate, the significance of differences between experimental groups was assessed using Student’s t-test. P10 nuclei (n=14)
9.7±0.7 8.8±0.8
26.7±5.4 14.1±2.9
29.7±2.3 23.2±2.0
a The latency values include the delay caused by the perfusion system; b a single exponential plus constant was fitted to the declining phase of the calcium transients starting at least 20 s after the removal of ATP
Fig. 2A–C Changes in [Ca2+]i evoked by the addition of 180 µM ATP in mouse myotubes. A A typical response of a mouse myotube that had more than five central nuclei. B The presence of 10 µM TTX and 100 µM verapamil did not interfere with the ability of ATP to elicit a calcium transient. C Signals evoked by repeated application of ATP. The vertical calibration ([Ca2+]i) is the same for all traces; separate horizontal calibrations (time) are given for A–B and C Fig. 1A–E Changes in cytosolic [Ca2+]i elicited by the application of 180 µM ATP in human cultured skeletal muscle cells. Myotubes with more than five central nuclei were used. A Typical slow response displaying a monotonic rising phase. B A biphasic response showing an early fast and a late slow increase in [Ca2+]i. Neither the application of 20 µM D-tubocurarine (C), nor the presence of 10 µM tetrodotoxin (TTX) and 100 µM verapamil (D) interfered with the ability of ATP to elicit a calcium transient. E The removal of external calcium ([Ca2+]e) abolished the ATP-evoked signal. In this and in all subsequent figures the unlabelled horizontal lines below each transient show the duration of ATP application and are positioned at 50 nM [Ca2+]i. The vertical calibration is the same for all traces; separate horizontal calibrations (time) are given for A–D and E
The most important kinetic properties of the ATPevoked signals (from hereon the slow component) are presented in Table 1. The calcium transients were characterised by relatively long latencies and were resistant to TTX and to blockade of the nAchR or the voltagegated, L-type calcium channels. As illustrated in Fig. 1C, addition of 20 µM D-tubocurarine to the bathing medium did not prevent ATP from eliciting a calcium transient. Similarly, as shown in Fig. 1D, in the presence of 100 µM verapamil a large change in [Ca2+]i followed the addition of ATP. Similar results were obtained on 22 myotubes. The removal of calcium from the external milieu completely abolished the response to ATP, as demonstrated in Fig. 1E. The lack of response to the second ATP challenge was not due to full desensitisation of the
signalling pathway since upon the readdition of calcium to the external solution ATP was again capable of evoking a calcium transient. Nevertheless, the increase in [Ca2+]i was somewhat smaller during the second and all subsequent ATP applications in all cells tested, indicating either a slow desensitisation or a run-down of the signalling pathway. These results establish the presence of an ATP-induced calcium signalling pathway in cultured human skeletal muscle cells that critically depends on the presence of calcium in the external solution. The present study focused on the effects of ATP on human skeletal muscle cells in culture. Since the purinoreceptor-encoding gene has been described for rodents, too, and is similar to that in human skeletal muscle [29], we also conducted a few experiments on cultured mouse skeletal muscle cells. These results were similar to those described above and are summarised in Fig 2. The application of 180 µM ATP in the external solution caused a transient rise in [Ca2+]i (as in Fig. 2A) with an average amplitude of 256±26 nM. The effect of ATP was present in the presence of TTX and verapamil (Fig. 2B), indicating that the ATP-induced elevation in [Ca2+]i did not depend on the depolarisation of the cell membrane or on the opening of voltage-gated L-type calcium channels. Calcium transients evoked by repeated applications of ATP on the same cell had similar amplitudes (Fig. 2C), confirming that desensitisation did not play an important role in the signalling pathway.
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Fig. 3 Developmental changes in the calcium transients. Changes in [Ca2+]i in cultured muscle cells were elicited by exposure to 180 µM ATP, 20 µM acetylcholine (Ach) or by replacing 120 mM NaCl with KCl in the external solution
Changes in the calcium signals during maturation From the beginning of the experiments it was evident that the amplitude and the kinetics of the ATP-evoked responses depended on the stage of development of the myotubes rather than on the days spent in culture. The first column of Fig. 3 illustrates ATP evoked calcium transients from human cultures, characteristic for different developmental stages, starting from myotubes with less than five nuclei (first row) to myofibres (cells with peripheral nuclei; last row). For comparison changes in [Ca2+]i evoked by the addition of 20 µM Ach (middle column) or by exchanging 120 mM sodium for potassium in the external solution (right column) are also shown. The ATP-evoked calcium transients were relatively small both at early and at late stages of development, while myotubes with more than five central nuclei responded with large changes in [Ca2+]i. In contrast, the amplitude of both the Ach- and the potassium-evoked signals increased monotonically with cell maturation, being the largest in large myofibres. These observations are presented as averages in Fig. 4B for five consecutive stages of development. It is evident from the figure that the age dependence of the ATP-evoked calcium transients was bell-shaped, while that for the Ach- or potassium-evoked signals increased monotonically, reaching a maximum for myotubes with more than ten nuclei. Although myoblasts failed to respond to any challenge with Ach or potassium, ATP did induce a small elevation in [Ca2+]i in these cells. While the responsiveness to these various agonists was clearly dependent on cell maturation, the resting [Ca2+]i remained fairly constant in the examined period (Fig. 4A). The actual values varied between 81±6 and 91±2 nM, demonstrating that the cells maintained a low [Ca2+]i similar to that reported earlier for cultured [1, 6, 8] or adult skeletal muscle fibres [34].
Fig. 4 The value of the resting [Ca2+]i (A) and the amplitude of the calcium transients (B) in five consecutive stages of development. Calcium signals were elicited by the addition of ATP, Ach or potassium as in Fig. 2. Number of cells investigated is given in parentheses
Concentration/response curve for ATP The concentration dependence of the ATP-evoked calcium transients was assessed by applying the agonist in concentrations of 1-180 µM. Myotubes having more than five central nuclei, i.e. those displaying the largest response to ATP (Fig. 4B), were used. To avoid any interference from possible desensitisation, only the first ATPinduced elevation in [Ca2+]i was included into the analysis for any given cell. Figure 5A presents characteristic calcium transients in response to 30-s applications of ATP. The changes in [Ca2+]i caused by a given concentration of ATP were then averaged and plotted as a function of the concentration in Fig. 5B. The full response was reached at around 100 µM, while the half-maximal activating concentration was around 20 µM. Although the data points followed a saturation curve they were not fitted with the Hill equation. Due to the presumably present contribution from intracellular calcium release and influx through voltage-gated channels, the parameters of a Hill fit would not represent those of the putative ATP-binding site on its receptor.
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Fig. 5A, B Concentration dependence of the ATP-induced calcium transients. A Representative calcium transients elicited by the addition of 1, 10, 50, 100 and 180 µM ATP. B Concentration/response curve for ATP. The number of cells tested at each concentration is given in parentheses
Effects of ATP analogues on [Ca2+]i To address the pharmacology of the ATP-induced influx pathway various analogues of ATP were tested for their ability to reproduce the effects of ATP. Figure 6A demonstrates that both α,β- and β,γ-methylene-ATP were much less effective than ATP itself. At 180 µM, α,βmethylene-ATP failed to induce any measurable change in [Ca2+]i in 7 out of 12 trials (58%), as depicted in Fig. 6A. Although β,γ-methylene-ATP was more effective, 17 out of 20 cells responded to the challenge, the amplitude of the calcium transient was still much smaller than that evoked by the subsequent addition of ATP. Figure 6A also demonstrates that these 25- and 15-s applications of α,β-and β,γ-methylene-ATP did not cause significant desensitisation of the ATP-induced signalling pathway. Although 2′,3′-O-(4-benzoyl)-benzoyl-ATP (BzATP) was more potent than the other ATP analogues, it was still not as potent as ATP (Fig. 6B). On average, the 180 µM BzATP-evoked signals had half the amplitude of those induced by 180 µM ATP (Fig. 6C). These values were 21% and 5% for β,γ- and α,β-methylene-ATP, respectively (Fig. 6C). At a lower concentration (10 µM), the difference between the effects of ATP and the studied analogues was somewhat more pronounced (Fig. 6C). ATP-evoked inward currents To ensure that the ATP-evoked changes in [Ca2+]i were indeed linked to the activation of a P2X receptor
Fig. 6A–C Calcium transients elicited by ATP analogues. A Small response to both α,β- and β,γ-methylene-ATP (180 µM). B Response to 2′,3′-O-(4-benzoyl)-benzoyl-ATP (BzATP). The vertical calibration is the same for A and B. C The relative effectiveness of the various analogues. The amplitudes of the calcium transients were normalised to those evoked by ATP at the same concentration in the given series of experiments. The number of cells is given in parentheses
activation, membrane currents were recorded in the absence and presence of 180 µM ATP. Cells were voltage clamped to –80 mV and were held at this potential to exclude the activation of any voltage-dependent ionic currents. As demonstrated in Fig. 7, a 60-s application of ATP induced an inward current, seen as a change in the holding current necessary to maintain the given holding potential. The average amplitude of this current was –260±25 pA (n=24). The currents had a characteristic rise time of 380±30 ms and were present with nearly constant amplitude during the application of the drug. After ATP removal the holding current returned to its original level. Repeated applications of ATP gave essentially identical current amplitudes, as depicted in the inset in Fig. 7, demonstrating that the ATP-evoked influx pathway showed little, if any, desensitisation.
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Fig. 7A, B ATP-induced inward currents on a myotube with five central nuclei. The holding potential was –80 mV and was held constant during the experiment. A Representative current record in response to a 60-s application of 180 µM ATP (horizontal calibration 50 s). Cell capacitance was 156 pF. B A part of the transient presented in A, on a different time scale (horizontal calibration 500 ms), to show the rising phase of the current. The inset shows the peak amplitudes of currents elicited by successive additions of ATP on the same cell. Similar results were obtained in seven cells
skin keratinocytes [31], this signal is thought to be one of the major components that initiates cell proliferation and differentiation and, ultimately, the formation of new skin. Although a similar role for ATP has not yet been demonstrated in skeletal muscle, altered expression of the human P2X receptor gene in certain degenerative or hyperproliferative muscle disorders has been suggested [37]. Furthermore, a mild degenerative muscle disease, sarcoglycanopathy, is associated with the loss [2] of an ecto-ATPase on the surface of the skeletal muscle fibres [13]. Any decrease in ecto-ATPase activity might cause accumulation of extracellular ATP resulting in a pathological activation of the purinergic signalling pathway. These observations also suggest a possible involvement of external ATP in the regeneration-differentiation process of human skeletal muscle. From the experiments presented here (Figs. 2, 3 and 4) and in the accompanying paper [10], it is likely that mature skeletal muscle fibres in adults have little, if any, sensitivity to ATP. Developing skeletal muscle cells or satellite cells, on the other hand, respond with changes in [Ca2+]i or with the development of an inward current as described for chick embryonic muscle [21]. It is of special interest that very early in muscle development, when neither Ach nor depolarisation induced by potassium elicited a calcium transient, myoblasts were already responsive to ATP. Since a small increase in [Ca2+]i is needed for the initiation of myoblast fusion [12] the ATP-dependent pathway could be a likely candidate. Source of calcium in the ATP-evoked calcium transients
Discussion The results demonstrated the ability of ATP to elevate [Ca2+]i in human and mouse skeletal muscle cells in culture. The amplitude of the calcium transients increased with cell maturation up to the point at which multinucleated myotubes form, then decreased. The transients displayed a variety of kinetics; from a slow monotonic rise to a biphasic behaviour in which the first, fast component was followed by a more gradual secondary increase. These effects of ATP were not affected by the nAChR antagonist D-tubocurarine but were mimicked by non-hydrolysable ATP analogues. ATP evoked a nondesensitising inward current with a rise time of around 300 ms and which could explain all the above mentioned effects of external ATP on [Ca2+]i. The observations are thus consistent with a functional purinergic, P2X signal transduction pathway in developing human skeletal muscle cells. Possible role of external ATP in muscle development The appearance of ATP in the extracellular space is associated with tissue damage but can also be the consequence of strenuous exercise [14]. In certain cells, e.g. in
Multi-nucleated myotubes often displayed biphasic calcium transients. A similar kinetic pattern has been described in cultured rat skeletal muscle cells for potassium-induced changes in [Ca2+]i [9]. This observation was interpreted as an activation of, and influx through, voltage-gated calcium channels triggered by the depolarisation and the subsequent calcium-induced calcium release from the sarcoplasmic reticulum (SR). Since in the present study the early fast component, seen mostly in large myotubes, was sensitive to TTX, we also believe that this fast component of the ATP-evoked signals represents depolarisation and the consequent activation of voltage-gated calcium channels. The slow component, on the other hand, could not simply reflect a calciuminduced calcium release mechanism, as suggested for rats [9], for to several reasons. Slow components were observed in the absence of the fast component in the majority (86%) of the cells, clearly indicating that the fast component was not a prerequisite for the presence of the slow. The fact that the removal of external calcium abolished the slow component suggests that the influx of calcium played an essential role in the initiation of the slow component. These data, however, do not rule out the possibility that calcium released from the SR participates in the slow component. In fact, influx of calcium through P2X purinoreceptors has been sug-
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gested to initiate calcium-induced calcium release in human smooth muscle cells [27]. Furthermore, ATP increases the production of inositol trisphosphate (IP3) in cultured chick [19] and rat myotubes [24]. Since IP3-receptors are present on the SR of developing human skeletal muscle cells [26] their opening could also contribute to the calcium signals. If SR calcium release were to have played an important role in the calcium transients seen here, it could explain the increase in the amplitude observed during the early stages of development, since both the size and the amount of calcium stored in the SR increases with cell differentiation [17, 23]. The decline in amplitude in large myofibres should then reflect a dramatic decrease in the number or sensitivity of the ATP receptors since the SR should be even more developed at this stage of differentiation. This would be consistent with findings in chick embryos in which a dramatic decline in responsiveness to ATP occurs between embryonic days 13–17 [38]. At this point one can only speculate on which of the P2X receptor subtypes could be serving as an influx pathway for calcium. The P2X receptor gene found in human skeletal muscle (P2XM) is distinct from all seven described previously [37]. Similarly, a novel P2X receptor subtype (chick P2X8) was cloned recently from chick skeletal muscle [4]. On the other hand, both P2X5 and P2X6 receptors have been demonstrated immunohistochemically in developing chick skeletal muscle [28]. Fitting the concentration/response relationship presented in Fig. 5B (not shown) gave a dissociation constant of 16 µM with a Hill coefficient of unity, implying the presence of P2X2, P2X4 or P2X5 receptors. These values should, however, be treated with caution. They probably do not reflect the actual parameters of the ATPbinding site since the amplitude of the calcium transient is influenced not only by the activation of the putative P2X receptor but also by the release of calcium from the SR and the removal of calcium from the myoplasmic space. Nevertheless, the observed order of potency for ATP analogues and the lack of desensitisation of the inward current are also consistent with P2X2, P2X4 or P2X7 subtypes [30, 33, 35]. On the other hand, the pharmacology of the P2XM receptor sub-type has not been documented so far, its presence cannot thus be excluded on the basis of these data. Acknowledgements The authors wish to thank Mrs. I. Varga and Ms. R. Öri for their excellent technical help. The work was funded by OTKA 030246, OTKA 034894, AKP 98-75 3,2/44, ETT 495/2000.
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