Abstract
Ca2+ oscillations are one of the most important signals within the cell. The mechanism for generation of Ca2+ oscillations is still not yet fully elucidated. We studied the role of capacitative Ca2+ entry (CCE) on intracellular Ca2+ oscillations induced by testosterone at the single-cell level in primary myotubes. Testosterone (100 nM) rapidly induced an intracellular Ca2+ rise, accompanied by Ca2+ oscillations in a majority of myotubes. Spectral analysis of the Ca2+ oscillations revealed a periodicity of 20.3 ± 1.8 s (frequency of 49.3 ± 4.4 mHz). In Ca2+-free medium, an increase in intracellular Ca2+ was still observed, but no oscillations. Neither nifedipine nor ryanodine affected the testosterone-induced Ca2+ response. This intracellular Ca2+ release was previously shown in myotubes to be dependent on inositol-1,4,5-trisphosphate (IP3). Intracellular Ca2+ store depletion in Ca2+-free medium, using a sarcoplasmic/endoplasmic reticulum calcium ATPase-pump inhibitor, followed by re-addition of extracellular Ca2+, gave a fast rise in intracellular Ca2+, indicating that CCE was present in these myotubes. Application of either testosterone or albumin-bound testosterone induced Ca2+ release and led to CCE after re-addition of Ca2+ to Ca2+-free extracellular medium. The CCE blockers 2-aminoethyl diphenylborate and La3+, as well as perturbation of the cytoskeleton by cytochalasin D, inhibited testosterone-induced Ca2+ oscillations and CCE. The steady increase in Ca2+ induced by testosterone was not, however, affected by either La3+ or cytochalasin D. These results demonstrate testosterone-induced Ca2+ oscillations in myotubes, mediated by the interplay of IP3-sensitive Ca2+ stores and Ca2+ influx through CCE.
Introduction
Testosterone, an anabolic steroid hormone, produces both genomic (Powers & Florini 1975, Mooradian et al. 1987) and non-genomic (Estrada et al. 2003) effects in skeletal muscle cells. The genomic effects involve interactions of testosterone with intracellular androgen receptor (Cato & Perterziel 1998, Lee et al. 2003), whereas non-genomic effects are characterized by rapid second messenger participation. We have shown that in myotubes testosterone produces an intracellular Ca2+ increase with an oscillatory pattern. The Ca2+ increase elicited by androgens in myotubes is due to Ca2+ released from intracellular stores, as the response is still produced in Ca2+-free medium. It involves the formation of inositol-1,4,5-trisphosphate (IP3) through phospholipase C activation because inhibitors of the IP3-mediated pathway almost completely inhibit the testosterone-induced Ca2+ increase (Estrada et al. 2000, 2003). The rapid effects of androgens are initiated at the plasma membrane, as shown by testosterone covalently bound to albumin (T-BSA), which does not cross the membrane yet still produces an intracellular Ca2+ increase which involves a receptor coupled to a pertussis toxin-sensitive G protein (Lieberherr & Grosse 1994, Benten et al. 1999, Estrada et al. 2003, Zagar et al. 2004). Thus, androgen-evoked Ca2+ oscillations in myotubes may involve concerted actions between events at the plasma membrane and internal stores, Ca2+ release from the sarcoplasmic reticulum being a key event for this mechanism. It has been proposed that different intracellular Ca2+ oscillatory patterns may preferentially activate or inactivate separate Ca2+-dependent processes related to regulation of gene expression (Li et al. 1998, Estrada et al. 2000, Powell et al. 2001). In myotubes, this could lead to, for example, differential activation or repression of genes due to Ca2+ signaling (Cato & Peterziel 1998, Jaimovich et al. 2000, Powell et al. 2001). In several cell types, agonist stimulation leads to a complex intracellular Ca2+ signal consisting of a peak increase due to the release of Ca2+ from the IP3-sensitive endoplasmic reticulum followed by a sustained phase due to the entry of Ca2+ from the external medium through store-operated channels (SOCs) (Putney et al. 2001, Venkatachalam et al. 2002). This secondary influx of Ca2+ is stimulated by the depletion of intracellular Ca2+ stores and has been called capacitative Ca2+ entry (CCE). Diverse hypotheses to explain the initiation of CCE have been suggested, including a diffusible factor, exocytosis of Ca2+-release activated Ca2+ channel and a structural link between the plasma membrane and intracellular stores. This last hypothesis involves a conformational coupling between IP3 receptors (IP3R) and Ca2+ channels in the plasma membrane (Venkatachalam et al. 2002). The existence of CCE in skeletal muscle cells has been demonstrated (Kurebayashi & Ogawa 2001, Islam et al. 2002), and conformational coupling between the plasma membrane and either ryanodine receptors (Islam et al. 2002) orIP3Rs (Kiselyov et al. 1998, Launikonis et al. 2003) has been postulated. In both models, it has been suggested that regulation of Ca2+ depletion from intra-cellular stores and the subsequent Ca2+ influx from the extracellular space through SOCs, requires an integral link between the plasma membrane and internal stores.
Actin microfilaments represent the main cytoskeletal component of differentiated skeletal muscle cells. It has been demonstrated that disruption of the actin cyto-skeleton in some cell types can affect the link between plasma membrane Ca2+ channels and intracellular Ca2+ stores (Pedrosa-Ribeiro et al. 1997) as well as affecting intracellular Ca2+ oscillations (Sergeeva et al. 2000). This effect depends on the cell type studied, as some reports indicate that disruption of actin filaments modifies the initial Ca2+ increases without affecting CCE (Pedrosa-Ribeiro et al. 1997), whereas others have shown that treatment with the fungal toxin cytochalasin D modifies both the Ca2+ increase in response to thapsigargin as well as subsequent CCE (Sergeeva et al. 2000). In this work we show that testosterone induces CCE in myotubes and that extracellular Ca2+ influx participates in testosterone-induced intracellular Ca2+ oscillations.
Material and Methods
Chemical reagents
Testosterone (4-androsten-17β-ol-3-one), T-BSA (testosterone-3-(o-carboxymethyl)oxime:BSA), thapsigargin, nifedipine and cytochalasin D were purchased from Sigma. 2-Aminoethyl diphenylborate (2-APB) was obtained from Aldrich (St Louis, MO, USA). Fluo-3 acetoxymethylester (Fluo-3 AM) was purchased from Molecular Probes (Eugene, OR, USA). Other reagents were of analytical grade.
Cell culture
Rats were bred in the Animal Breeding facility of the Faculty of Medicine, University of Chile. We performed all studies with the approval of the institutional bioethical committee. Rat myotubes were cultured as reported previously (Estrada et al. 2000, Jaimovich et al. 2000). Briefly, myoblasts were obtained from neonatal rat hind limbs. The tissue was mechanically dispersed and then treated with 10% (w/v) collagenase for 15 min at 37 °C under mild agitation. The suspension was filtered through a Nytex (Sartorius, Goettingen, Germany) membrane and spun down at low speed. Pre-plating was used to partially eliminate fibroblasts; cells were then plated onto round coverslips at a density of ~3.5×105 per dish. The culture medium was DMEM/F-12 without phenol red, 10% bovine serum, 2.5% fetal calf serum, 100 mg/l penicillin, 50 mg/l streptomycin and 2.5 mg/l amphotericin B. To eliminate remaining fibroblasts, 10 μM cytosine arabino-side was added on the third day of culture for 24 h. The medium was then replaced with serum-free medium. Myotubes with an estimated purity of more than 90% were visible after the fifth day of culture. Unless otherwise indicated, we used 6- to 8-day-old cultures exhibiting a fairly homogeneous population of myotubes with central nuclei, measuring 200–300 μm long and 20–40 μm wide.
Intracellular Ca2+
For intracellular Ca2+ measurements at the single-cell level, myoblasts were cultured on glass coverslips to 80% confluence and then differentiated into myotubes by withdrawal of serum. Ca2+ images were obtained from myotubes loaded with the fluorescent Ca2+ dye Fluo-3 AM using an epifluorescence microscope (T041; Olympus Corp., New Hyde Park, NY, USA) equipped with a cooled CCD camera and image acquisition system (MCD 600; Spectra Source Instruments, Westlake Village, CA, USA). Myotubes were washed three times with Krebs buffer (145 mM NaCl, 5 mM KCl, 2.6 mM CaCl2, 1 mM MgCl2, 10 mM Hepes-Na, 5.6 mM glucose, pH 7.4) to remove serum, and loaded with 5.4 μM Fluo-3 AM (coming from a stock in pluronic acid–dimethylsulfoxide 20%) for 30 min at room temperature. After loading, myotubes were washed with Krebs buffer for 10 min to allow the de-esterification of the dye, and used within 2 h. The coverslips were mounted in a 1 ml capacity plastic chamber and placed on the microscope for fluorescence measurements. Fluorescence images were collected every 1.0–2.0 s and analyzed frame by frame with the data acquisition program of the equipment (MCD 600; Spectra Source). A PlanApo 40 × (NA 1.4) objective lens was used. In most of the acquisitions, the image dimensions were 512×120 pixels. Inhibitors were added during the dye incubation; times and concentrations are indicated in the results section. To assess the role of the actin cytoskeleton on the intracellular Ca2+ response, cytochalasin D was added 20 min or 1 h before hormone stimulation. Intracellular Ca2+ was expressed as a percentage of fluorescence intensity relative to basal fluorescence (a value stable for at least 5 min in resting conditions). The increase in fluorescence intensity of Fluo-3 AM is proportional to the rise in intracellular Ca2+ (Minta et al. 1986). Each experiment involved a single independent cell and whole cell fluorescence was acquired. A given cell was considered to oscillate when oscillations were evident in the whole cell record.
Digital image processing
Elimination of out-of-focus fluorescence was performed by software. Both the ‘no-neighbors’ deconvolution algorithm and Castleman’s point spread function theoretical model were used. Complementary to restoration methods, a procedure was created to section the images. To segment an image, an initial contour can be entered manually, and a recursive algorithm that adapts automatically to the region of interest (adaptable contour) can be applied (Estrada et al. 2000). To quantify fluorescence, the summed pixel intensity was calculated for the section delimited by a contour. As a way of increasing the effciency of these data manipulations, action sequences were generated. To avoid possible interference in the fluorescence by changes in volume after exposure to steroids, the area of a fluorescent cell was determined by image analysis using an adaptive contour and then creating a binary mask, which was compared with a bright-field image.
Power spectrum analysis
We used an algorithm written in MATLAB to perform power spectrum analysis. The power spectrum of a signal is the squared Fourier transform, and describes the contribution to that signal by each of its sine wave components. The oscillating section from a single cell measurement was filtered, centered and trend corrected by computing the Gauss least-square approximation. To derive the discrete Fourier transform, a fast Fourier transform was used. This calculation produced a spectrum where the peaks correspond to the different frequencies present in the original data. The most dominant peak was determined by comparing the relative power of the peaks in the spectrum. The relative power was determined by calculating the area between the two extremes closest to the peak and dividing by the total area of the power spectrum, as described (Aizman et al. 2001, Miyakawa-Naito et al. 2003).
Statistics
Differences between basal and post-stimulated points were determined using a paired Student’s t-test. P < 0.05 was considered statistically significant.
Results
We have previously demonstrated that testosterone induces intracellular Ca2+ increases independently of the intracellular androgen receptor (Estrada et al. 2000, 2003). From those studies, a concentration of 100 nM testosterone was determined to produce highly reproducible Ca2+ responses in myotubes and so was used for the following set of experiments. The effects of testosterone (100 nM) on intracellular Ca2+ in myotubes are shown in Fig. 1A. From a total of 176 cells in 32 independent primary cultures, 134 cells responded to testosterone with a Ca2+ increase. A majority of cells challenged with testosterone exhibited Ca2+ oscillations (76%; n=101 of 134 cells). This oscillatory response was initiated by a rapid peak in the intracellular Ca2+ accompanied by oscillations. When the cells were exposed to testosterone, Ca2+ rapidly increased (34 ± 12 s range 10–49 s) after hormone addition. This Ca2+ rise was maintained for 1–2 min while the oscillatory pattern was observed. Once the intracellular Ca2+ concentration returned to the basal level, oscillations could no longer be detected. Responding cells that did not oscillate exhibited a similar rise in intracellular Ca2+. Ca2+ oscillations are characterized by frequency and amplitude. These two features have previously been shown to be of critical importance for the physiological response activated by Ca2+ oscillations (Dolmetsch et al. 1998, Li et al. 1998). To examine the regularity of testosterone-induced Ca2+ oscillations we performed a power spectrum analysis (Fig. 1B). By applying this method we could determine similarities among all single-cell recordings. The power spectrum of an oscillatory signal describes the contribution to that signal of different sine wave components (Aizman et al. 2001). This analysis thus allows the most dominant frequency contribution to be determined from a complex signal. Moreover, irregular and random contributions to the signal are excluded by this approach. Spectral analysis of Ca2+ oscillations induced by testosterone generated a signal which could be described with an average frequency of 49.3 ± 4.4 mHz, which corresponds to an average periodicity of 20.3 ± 1.8 s.
In order to determine the source(s) of Ca2+ implicated in the testosterone-induced Ca2+ increase, myotubes were incubated in Ca2+-free medium (1 mM EGTA) prior to androgen stimulation. Under this condition, a sustained Ca2+ increase was still seen for both testosterone-treated (Fig. 2A; n=32 of 41 cells; seven independent cultures) and T-BSA-treated cells (Fig. 2A, inset; n=12 of 14 cells; five independent cultures); however, intracellular Ca2+ oscillations were completely abolished. These results suggest that the Ca2+ response elicited by testosterone consists of at least two components: an intracellular release contributing to the rise in Ca2+, and a Ca2+ influx from the extracellular medium, which is required for oscillations. We have previously shown that Ca2+ signals evoked by testosterone were dependent on both the generation of IP3 and on the activity of IP3Rs (Estrada et al. 2000, 2003). We now tested whether Ca2+ signals were also related to ryanodine and dihydropyridine receptors. Oscillations were not due to activation of L-type voltage-operated Ca2+ channels, as nifedipine (10 μM) did not modify the Ca2+ oscillations induced by testosterone (Fig. 2B; n=12 of 16 cells; three independent cultures). Similarly, pre-treatment of myotubes with 20 μM ryanodine, a concentration known to block Ca2+ release through ryanodine receptors (Jaimovich et al. 2000), did not modify the hormone-mediated intracellular Ca2+ oscillations (Fig. 2B; n=10 of 14 cells; three independent cultures), suggesting that Ca2+ mobilization did not involve ryanodine receptors.
Treatment of myotubes with thapsigargin (1 μM), an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase pump, in Ca2+-free medium showed an intracellular Ca2+ increase due to Ca2+ release from intracellular stores (Fig. 3A; n=20 of 20 cells; four independent cultures). A slow Ca2+ increase followed by a decrease back to the basal level characterized this response. The intracellular Ca2+ level of myotubes not treated with thapsigargin was unchanged after the removal of Ca2+ in the extracellular medium (n=12 of 12 cells; three independent cultures, data not shown). Re-addition of Ca2+ (2 mM) to the extracellular medium produced a fast intracellular Ca2+ increase in thapsigargin-treated cells, suggesting that depletion of intracellular stores promoted Ca2+ entry from the extracellular medium (Fig. 3A).
These results suggest that activation of CCE occurs by emptying the IP3-sensitive Ca2+ stores according to a previously demonstrated testosterone-induced IP3 activation pathway (Estrada et al. 2000, 2003).
Pre-treatment of myotubes with thapsigargin blocked the Ca2+ signal induced by testosterone, indicating that the Ca2+ increase produced by this hormone involves, at least in part, thapsigargin-sensitive intracellular Ca2+ stores (Fig. 3B; n=8 of 8 cells; three independent cultures). Both La3+, a non-specific Ca2+ channel blocker, and 2-APB, were reported to inhibit CCE in several cellular models (Jaimovich et al. 2000, Bootman et al. 2002, Collet & Ma 2004). In accord with these reports, La3+ and 2-APB did not inhibit thapsigargin-induced Ca2+ release from intra-cellular stores (Fig. 3C). The thapsigargin-evoked Ca2+ entry in myotubes was, however, significantly reduced by 1 μM to 1 mM La3+ (80%, P < 0.05; n=16 of 16 cells; five independent cultures) or 50 μM 2-APB (76%, P < 0.05; n=18 of 18 cells; six independent cultures) (Fig. 3C).
To determine whether CCE participates in the response to testosterone, experiments similar to those with thapsigargin were performed. Figure 4A shows a testosterone-induced intracellular Ca2+ increase in a myo-tube incubated in Ca2+-free medium. Re-addition of 2 mM Ca2+ to the extracellular medium produced a rapid and sustained Ca2+ entry (Fig. 4A; n=21 of 21 cells; four independent cultures). These results suggest that activation of CCE occurs by emptying the IP3-sensitive Ca2+ stores according to a testosterone-induced IP3 activation pathway previously demonstrated (Estrada et al. 2000, 2003). The vehicle, ethanol ( < 0.01%), did not induce a Ca2+ increase and re-addition of extracellular Ca2+ (2 mM) produced only a slight increase in intracellular Ca2+ (Fig. 4C; ΔF/F=5.2 ± 3.1; n=6 of 6 cells; two independent cultures). To verify that the testosterone-induced Ca2+ release was not due to activation of the intracellular androgen receptor, we performed experiments using plasma-membrane-impermeable T-BSA. Under similar conditions, T-BSA induced CCE (Fig. 4B; n=12 of 12 cells; four independent cultures) but albumin by itself did not cause any intracellular Ca2+ increase (Fig. 4D; n=6 of 6 cells; two independent cultures). In these myotubes the Ca2+ re-addition protocol produced a relative fluorescence increase of 6.1 ± 1.1. This small rise in the baseline Ca2+ signal after re-addition of extracellular Ca2+ in vehicle-treated cells could be expected, in Ca2+-free conditions, through cytosolic Ca2+ leak pathways more than through activation of a CCE pathway. The relative change of fluorescence intensity was at least one order of magnitude greater when cells were exposed to testosterone vs vehicle, which suggests that Ca2+ entry is activated by testosterone application.
To determine whether CCE participates in the generation of testosterone-induced Ca2+ oscillations, experiments using CCE inhibitors were performed. As shown in Fig. 5A, application of La3+ blocked testosterone-evoked Ca2+ oscillations. Pre-incubation of myotubes with La3+ before the addition of testosterone in Ca2+-free medium did not cause a detectable inhibition of testosterone-induced Ca2+ release from intracellular stores, but completely inhibited Ca2+ oscillations (Fig. 5B; n=11 of 11 cells; four independent cultures). Moreover, Fig. 5B shows that La3+ and 2-APB inhibited steroid-induced Ca2+ influx in Ca2+ re-addition experiments by 80% (range 68–95%, P < 0.05; n=18 of 18 cells; four independent cultures) and by 73% (range 65–82%, P < 0.05; n=25 of 25 cells; seven independent cultures) respectively, similar to effects of these agents on thapsigargin-induced Ca2+ influx (Fig. 3C). These results suggest that CCE participates in testosterone-induced Ca2+ oscillations in rat skeletal myotubes.
To assess the role of the actin cytoskeleton on intracellular Ca2+ increases induced by testosterone, cytochalasin D, an actin-depolymerizing agent, was applied to disrupt the cytoskeletal structure of actin filaments. Pre-incubation of myotubes with 10 μM cytochalasin D for 20 min prior to testosterone application reduced the number of oscillating cells (30% vs 76% of testosterone-responsive cells; n=21 cells from four independent cultures), whereas pre-incubation for 1 h blocked Ca2+ oscillations (Fig. 6A; n=18 of 18 cells; three independent cultures). The testosterone-induced intracellular Ca2+ increase persisted, although reduced in magnitude (29% with respect to control (Fig. 2A), P < 0.05; range 15–38; n=20 of 20 cells; three independent cultures). The role of the actin cytoskeleton on CCE mediated by testosterone was also evaluated through re-addition of extracellular Ca2+. Figure 6B shows that incubation of myotubes with cytochalasin D for 1 h reduced the testosterone-stimulated Ca2+ rise in Ca2+-free medium, and inhibited testosterone-induced CCE by 43% (range 23–56%, P < 0.05; n=10 of 10 cells; two independent cultures) with respect to the control condition. These results imply that an intact actin cytoskeleton is necessary for myotubes to generate Ca2+ oscillations.
Discussion
Androgens can produce rapid effects in myotubes through mechanisms other than a genomic response; however, these mechanisms are not yet fully understood. Previously, we demonstrated that a key step in the rapid testosterone response of myotubes is an increase in intracellular Ca2+, observable as Ca2+ oscillations and propagated Ca2+ waves (Estrada et al. 2000, 2003). In this study, we show evidence that testosterone-induced Ca2+ responses in myotubes are complex, involving both intracellular and extracellular Ca2+ and can be divided into two components: Ca2+ release from IP3-sensitive stores (Estrada et al. 2000, 2003), which causes a rapid intracellular Ca2+ increase, and a CCE pathway through the plasma membrane, responsible for Ca2+ oscillations. Both these mechanisms are essential for the testosterone-induced Ca2+ response in myotubes.
In myotubes, the long-lasting Ca2+ rise of testosterone-induced signaling is produced in Ca2+-containing as well as Ca2+-free medium, suggesting Ca2+ mobilization from internal stores, consistent with previous findings using inhibitors of IP3-mediated Ca2+ pathways such as U73122 and xestospongin B (Estrada et al. 2003). These results strongly suggest that stimulation of myotubes with testosterone induces an intracellular Ca2+ increase through phosphoinositide signaling pathways. Intracellular Ca2+ oscillations are a common event in many different cell types. Different oscillatory patterns suggest different mechanisms of Ca2+ release and re-uptake as well as different signaling functions for intracellular Ca2+ (Dolmetsch et al. 1998, Li et al. 1998, Sneyd et al. 2004). In a high percentage of cells studied, the testosterone-induced Ca2+ rise was accompanied by Ca2+ oscillations, which may represent an important early step for the coordination of cell functions in skeletal muscle (Shtifman et al. 2004). In this study we show that the oscillatory pattern induced by testosterone exhibits a remarkably constant frequency (49.3 ± 4.4 mHz) corresponding to a periodicity of ~20 s, suggesting a highly regulated event. Interestingly, oscillations tend to decrease and fade 2 min after testosterone stimulation. This is consistent with the transient increase of IP3 seen after testosterone addition, which returns to basal values after 2 min (Estrada et al. 2003). Collet & Ma (2004) have proposed a regulatory mechanism for CCE in skeletal muscle with an enhancement of SOC activity upon initial entry of extracellular Ca2+ followed by gradual and complete deactivation of the SOC channel function associated with the uptake of Ca2+ into the sarcoplasmic reticulum, which represents a graded deactivation process for CCE regulation, through Ca2+ storage, in times compatible with our results. Rapid frequency-dependent signals can be used by cells to activate simultaneously several cellular processes, thus allowing the same second messenger to be used for several different events. It has been reported that specific frequencies can activate specific genes (Dolmetsch et al. 1998, Li et al. 1998). Ca2+ oscillations have been shown in several biological systems. This study shows that a hormone, testosterone, can induce Ca2+ oscillations of a specific frequency. Testosterone-evoked Ca2+ oscillations only occurred in the presence of extracellular Ca2+. In several cell models, Ca2+ oscillations are reported to be initiated by IP3-induced release of Ca2+ from intracellular Ca2+ stores (Berridge & Irvine 1989, Aizman et al. 2001). They are dependent, however, on Ca2+ influx through Ca2+ channels in the plasma membrane (Berridge & Irvine 1989, Sneyd et al. 2004). Ca2+ oscillations induced by testosterone stimulation in myo-tubes thus appear to be similar to agonist-evoked Ca2+ oscillations in other excitable and non-excitable cells (Berridge & Irvine 1989, Sergeeva et al. 2000, Aizman et al. 2001, Sneyd et al. 2004).
Depletion of intracellular Ca2+ stores by thapsigargin promoted activation of Ca2+ entry from the extracellular medium, suggesting the presence of a CCE pathway. Treatment of myotubes with thapsigargin blocked the Ca2+ signal induced by testosterone, indicating that the Ca2+ increase produced by this hormone involved intracellular Ca2+ stores sensitive to thapsigargin. Moreover, the fast Ca2+ entry after Ca2+ re-addition experiments in myotubes stimulated by testosterone or T-BSA in Ca2+-free medium indicates that testosterone activates a plasma membrane Ca2+ influx. The existence of CCE in skeletal muscle cells involving conformational coupling between the plasma membrane and either ryanodine receptors (Islam et al. 2002) or IP3Rs (Launikonis et al. 2003) has been postulated. We have previously shown that there is a caffeine-sensitive Ca2+ pool in these cells (Carrasco et al. 2003). In this study, however, ryanodine did not inhibit Ca2+ oscillations, suggesting that these effects are dependent on IP3R activation. All three types of IP3R have been found to be present in myotubes (C Cárdenas, J L Liberona, J Molgó, C Colasante, G A Mignery & E Jaimovich, unpublished observations). Launikonis et al.(2003) have demonstrated in mechanically skinned skeletal muscle cells that IP3R mediates SOCs, and show evidence that the IP3R can act as a sarcoplasmic reticulum Ca2+ sensor necessary for CCE. It has further been suggested that IP3R could be physically coupled to integral membrane proteins, such as SOCs (Kiselyov et al. 1998, Ma et al. 2000, Launikonis et al. 2003) or Na+, K+-ATPase (Miyakawa-Naito et al. 2003). In adult skeletal muscle, CCE (through SOCs) was insensitive to nifedipine (Kurebayashi & Ogawa 2001). In contrast, the role of voltage-gated Ca2+ channels in CCE was suggested in other cell types (Densmore et al. 1996, Aizman et al. 2001) and steroid-induced CCE has been postulated to occur through a transient receptor potential protein channel 3 (TRPC3)-like protein in rat osteoblasts (Baldi et al. 2003). Spontaneous Ca2+ oscillations in myotubes were described by Shtifman et al.(2004). These oscillations were inhibited by Cd3+/La3+, but also by nifedipine and so were attributed to Ca2+ entry through L-type Ca2+ channels. In our study, Ca2+ entry triggered by testosterone in myotubes was insensitive to the voltage-dependent Ca2+ channel antagonist nifedipine, but was inhibited by 2-APB and the non-specific Ca2+ channel blocker La3+, as identified by inhibition of Ca2+ entry in the Ca2+ re-addition protocols. At the concentration used, 2-APB has been shown to be a blocker of SOCs in several cell types including skeletal muscle (Bootman et al. 2002, Collet & Ma 2004). Moreover, our results suggest that CCE participates in the generation of testosterone-induced Ca2+ oscillations, because both 2-APB and La3+ blocked this effect. 2-APB inhibited the testosterone-induced, IP3-dependent Ca2+ signal by 43% whereas the CCE signal was inhibited by more than 66%, indicating that CCE is more sensitive to this inhibitor than the IP3R pathway. Pre-incubation of myotubes with La3+ before the addition of testosterone, in Ca2+-free medium, did not cause a detectable inhibition of testosterone-induced Ca2+ release from intracellular stores, but completely inhibited Ca2+ oscillations.
Kiselyov et al.(1998) have suggested a physical interaction between IP3R and the plasma membrane that involves the actin cytoskeleton, and Mohler et al.(2004) have demonstrated a link between IP3R and ankyrin-B, a protein known to bind membrane proteins to the actin cytoskeleton, important in localization and stabilization of the receptor in neonatal cardiomyocytes. Cytochalasin D blocked intracellular Ca2+ oscillations, but not the testosterone-induced long-lasting rise in intracellular Ca2+. This drug only partly reduced the CCE seen upon re-introduction of Ca2+ to Ca2+-free external medium.
Early changes in myotubes by steroids could be directly related to activation of Ca2+-mediated events. The differential activation of a genomic or a non-genomic pathway could be important to the physiological relevance of testosterone in skeletal muscle, mediating such physiological responses as muscle hypertrophy. An early event in skeletal muscle hypertrophy is an increase in intracellular Ca2+ (Semsarian et al. 1999). Thus, for testosterone this mechanism is amenable to a two-step process as described for others steroid hormones (Wehling 1997), where both non-genomic and genomic effects occur sequentially. Thus, the pathways used by steroid hormones in different cell types could add another dimension of signal specificity. Ca2+ oscillations with frequency ranging from 10 to 50 mHz (periodicities from 20 to 100 s) have been described in various cell types upon different stimulation protocols (Li et al. 1998, Sneyd et al. 2004). Interestingly in cultured myotubes, spontaneous Ca2+ oscillations with a frequency of approximately 45 mHz were described (Shtifman et al. 2004).
Taken together, these observations provide functional evidence for the existence of CCE induced by testosterone, which is necessary for the generation of Ca2+ oscillations in myotubes. The response is complex and is mediated by interplay between IP3-sensitive Ca2+ stores (Estrada et al. 2000, 2003) and Ca2+ influx through voltage-independent channels activated by store depletion.
Funding
This work was financed by: FONDECYT Grants 2000-055 and 1030988 (M E), (P U) Vetenskapsrådet – the Swedish Research Council – and (C J G) Howard Hughes Medical Institute predoctoral fellowship. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
Intracellular Ca2+ oscillations induced by testosterone in myotubes. Testosterone (100 nM) induced a rapid intracellular Ca2+ increase with an oscillatory pattern in Fluo-3 AM-loaded myotubes. (A) A representative single-cell trace of myotube response in a Ca2+-containing medium, and (B) the corresponding power spectrum analysis of the oscillations from the same cell as in (A). In this particular experiment, the analysis reveals a relatively constant oscillation frequency of 41.1 mHz, equivalent to a period of 24.3 s. The arrow indicates the time of testosterone (T) addition. A.U., arbitrary units.
Citation: Journal of Endocrinology 184, 2; 10.1677/joe.1.05921
Effect of Ca2+-free medium, nifedipine and ryanodine on testosterone-induced Ca2+ oscillations. (A) Pre-incubation of Fluo-3 AM-loaded myotubes in Ca2+-free medium (1 mM EGTA) for 5 min did not affect the fluorescence rise after stimulation with 100 nM testosterone or T-BSA (inset), but did inhibit subsequent Ca2+ oscillations. (B) Representative traces of experiments in the presence of Ca2+ channels inhibitors. Neither nifedipine nor ryanodine modified the testosterone-induced Ca2+ oscillations in a Ca2+-containing medium.
Citation: Journal of Endocrinology 184, 2; 10.1677/joe.1.05921
Thapsigargin (Thapsi) activation of SOCs in myotubes. Cells loaded with Fluo-3 AM were incubated in Ca2+-free medium and treated with 1 μM thapsigargin. (A) In the absence of extracellular Ca2+, thapsigargin induced a transient increase in intracellular Ca2+. Subsequent addition of Ca2+ (2 mM) to the extracellular medium resulted in a large intracellular Ca2+ increase suggesting initiation of CCE. (B) Testosterone did not produce any change in the fluorescence in myotubes after depletion of thapsigargin-sensitive stores. (C) Pre-incubation with La3+ or 2-APB did not affect the thapsigargin-induced Ca2+ transient, but did reduce the subsequent intracellular Ca2+ rise after addition of 2 mM Ca2+ to the extracellular medium, further supporting the suggestion of thapsigargin-sensitive CCE.
Citation: Journal of Endocrinology 184, 2; 10.1677/joe.1.05921
Testosterone- and T-BSA-induced activation of CCE in myotubes. Cells bathed in Ca2+-free extracellular medium were stimulated with testosterone (A) or T-BSA (B). Both initiated a transient intracellular Ca2+ increase that was not seen when cells were stimulated with vehicle (C) or BSA alone (D). Upon re-addition of extracellular Ca2+ (2 mM), hormone-stimulated cells exhibited a sustained intracellular Ca2+ increase not present in control-stimulated cells, suggesting a mechanism of hormone-induced CCE.
Citation: Journal of Endocrinology 184, 2; 10.1677/joe.1.05921
Effect of La3+ and 2-APB on testosterone-induced Ca2+ signals. (A) Testosterone was added in the absence of external Ca2+. La3+ inhibited the Ca2+ oscillations but not the initial Ca2+ increase induced by testosterone. (B) Inhibitors of CCE were added 10 min before testosterone stimulation. Upon Ca2+ re-addition, CCE was reduced by either La3+ or 2-APB. These results suggest that testosterone activates CCE in a similar manner to thapsigargin (Fig. 3C), and that this CCE is required for testosterone-induced intracellular Ca2+ oscillations. The arrow indicates the time of addition of testosterone (T).
Citation: Journal of Endocrinology 184, 2; 10.1677/joe.1.05921
Ca2+ responses in cytochalasin D-treated myotubes induced by testosterone. Myotubes were treated with cytochalasin for 1 h and then loaded with Fluo-3 AM to detect changes in intracellular Ca2+. (A) Cytochalasin D inhibited the Ca2+ oscillations, but not the sustained Ca2+ increase, induced by testosterone. (B) The cytoskeletal disruption in cytochalasin D-treated cells reduced the testosterone-induced CCE by 43% (note that the values of relative fluorescence in control conditions are in the range of 120–180 (Figs 3, 4 and 6)). The arrow indicates the time of addition of testosterone (T).
Citation: Journal of Endocrinology 184, 2; 10.1677/joe.1.05921
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