Introduction

Ca2+ is a universal second messenger which triggers short term cellular responses (e.g. contraction in heart and skeletal muscle) as well as long term cellular adaptive responses (e.g. T-lymphocyte reactivity or long term potentiation in neuronal cells) (Berridge 1997; Berridge et al. 1998; Clapham 1995). The intracellular free Ca2+ concentration, [Ca2+]i, is modulated by two sources, influx from the extracellular space and release from intracellular Ca2+ stores. In both cases specific Ca2+ channels mediate the transmembraneous Ca2+ fluxes.

Putney proposed for the first time that emptying of internal Ca2+ stores is linked to a so called store-operated Ca2+ influx from the extracellular space (Putney 1986). Currently, a variety of receptors have been found to couple intracellular Ca2+ release via the InsP3 receptor to Ca2+ influx in excitable as well as non-excitable cells (Barrit 1999; Hofmann et al. 2000). Two major mechanisms are proposed to explain store-operated Ca2+ influx (Montell 2001; Putney and McKay 1999): first, a yet not identified Ca2+ influx factor is proposed which is thought to trigger Ca2+ influx from voltage-insensitive Ca2+ channels (Randriamampita and Tsien 1993; Rzigalinski et al. 1999). Secondly, Irvine proposed a conformational coupling between the inositol 1,4,5-triphosphate (InsP3) receptors and store-operated Ca2+ channels analogous to the conformational and functional coupling between the ryanodine receptors and L-type Ca2+ channels in skeletal muscle (Irvine 1990). The store operated Ca2+ current is in part thought to be mediated by ion channels of the transient receptor potential-canonical (TRPC) family. TRPC channels have been found to couple to both intracellular Ca2+ channels, the InsP3 receptor and the ryanodine receptor (Kiselyov et al. 1998, 2000; Rosado and Sage 2000). Human TRPC3 channels stably expressed in HEK293 cells were found to couple exclusively to either InsP3 receptors or ryanodine receptors (Kiselyov et al. 2000).

A direct contact between the α1S-subunit of the L-type Ca2+ channel and the skeletal muscle ryanodine receptor (RyR type 1) is a structural prerequisite for excitation-contraction (EC) coupling in skeletal muscle (Schneider 1994). Consequently, depolarisation is sufficient to trigger intracellular Ca2+ transients via the RyR type 1 in the absence of a detectable Ca2+ influx (O'Brian et al. 2002). The intracellular Ca2+ stores, namely the sarcoplasmic reticulum, have limited capacity for Ca2+ storage. Therefore, efficient refilling of these stores is essential for repeated cycles of muscle contraction and relaxation. The L-type Ca2+ channel has been proposed to compensate for this loss of Ca2+ but it is believed that Ca2+ channels which activate the RyR type 1 do not serve as Ca2+ conductors (O'Brian et al. 2002; Tanabe et al. 1990, 1991). That is why the voltage-insensitive Ca2+ influx may play a prominent role to supply the muscle cell with sufficient Ca2+ (Hopf et al. 1996a; Kurebayashi and Ogawa 2001).

Myotubes derived from human satellite cells are a prototypical system for investigation of RyR type 1 mediated Ca2+ transients. In the present work we have used differentiated human skeletal muscle cells as a native model system to investigate the linkage of the selective depletion of ryanodine sensitive Ca2+ pools to voltage insensitive Ca2+ influx. We used specific pharmacological tools to exclude the involvement of the phospholipase C products InsP3 and diacylglycerol in this store operated Ca2+ influx. We could also identify a diacylglycerol dependent Ca2+ influx which was strictly dependent on protein kinase C activity.

Materials and methods

Materials

Sera and media for cell culture were obtained from PAA (Linz, Austria), trypsin-EDTA, glutamine, penicillin, streptomycin, gentamicin and amphotericin B were obtained from GibcoBRL (Vienna, Austria), xestospongin C, 1,6-bis (cyclohexyloximinocarbonylamino) hexane (RHC-80267), 1,2-Dioctanoyl-sn-glycerol (DOG), 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3- (1H-indol-3-yl)-maleimide (GF109203X), staurosporine and nifedipine from Calbiochem (San Diego, CA, USA), aprotinin from Bayer AG (Wuppertal, Germany), Pefabloc from Boehringer Mannheim (Mannheim, Germany), fibronectin from Collaborative Biomedical Products (Bedford, MA, USA). All other chemicals were from Sigma-Aldrich (Vienna, Austria).

Cell culture

The study conformed to the code of Ethics of the World Medical Association (Declaration of Helsinki) and was approved by the Ethics Committee of the AKH Wien (University Hospital of Vienna). Patients, who underwent the in vitro contracture test to verify susceptibility for malignant hyperthermia (MH) gave informed written consent to use waste material from their muscle biopsies (200–400 mg) for muscle cell culture. The present study was carried out with cells from eight individuals, found to be MH non susceptible. Human skeletal muscle cells were prepared and cultured according to the method of Brinkmeier et al. (1993) and grown in growth medium (GM) or differentiation medium (DM) according to Baroffio et al. (1993). GM contained Ham's F12 supplemented with 15% foetal calf serum 10 ng/ml EGF, 200 ng/ml insulin, 400 ng/ml dexamethasone, 0.5 mg/ml fetuin, 0.5 mg/ml BSA, 7 mM glucose, 4 mM L-glutamine, 200 U/ml penicillin, 200 µg/ml streptomycin, 2.5 µg/ml amphotericin B. DM contained: DMEM supplemented with 5% horse serum, and 4 mM L-glutamine, 100 ng/ml insulin, 0.1 µg/ml gentamicin.

Cells were grown at 37°C under 2.5% CO2 close to confluency and reseeded on 25 mm glass cover slips coated with fibronectin for imaging experiments, or 75 cm2 flasks for mass cell fluorescence photometry. Thereafter adherent cells were switched to DM and 5% CO2 to obtain myotubes. Experiments were done within a period of 3 to 10 days after changing the culture medium from GM to DM.

Ca2+ imaging and fluorescence photometry

Ca2+ fluorescence imaging (Nikon Diaphot 300 fluorescence microscope) was carried out with myotubes incubated in 7 µM fura2/AM. Dye loading conditions were identical as previously described (Weigl et al. 2000). Fluorescence intensity was monitored at an emission wavelength of 510 nm with excitation by a monochromator at 340 and 380 nm (VisiTech, Sunderland, UK). Stored images were analysed using the QC 900 software package. Regions of interest were defined covering the whole visible area of a cell and the corresponding light intensity values were further processed with the Sigma Plot program (SPSS Inc., Erkrath, Germany). Background subtraction, ratioing and calculation of the intracellular Ca2+ concentration ([Ca2+]i) were done off-line. Calculation of [Ca2+]i values was carried out according to the equation of Grynkiewicz et al. (1985). The parameters for the equation were obtained by a calibration procedure in which we used the penta-potassium salt of fura2 (5 µM) in a solution mimicking the intracellular milieu (Thomas and Delaville 1991). The KD of Ca2+ for fura2 was assumed to be 224 nM. The fluorescence ratio in the absence of Ca2+ (Rmin) gave 0.39 and was 10.64 in the presence of saturating concentrations of Ca2+ (Rmax). The quotient (β) of the fluorescence values for Ca2+ bound and Ca2+ free dye at 380 nM was 10.75.

The quantification of the Ca2+ influx was obtained from subtracting basal Ca2+ concentrations after the drug application (caffeine or nifedipine) in the absence of extracellular Ca2+ from the peak Ca2+ concentration in the presence of extracellular Ca2+ (cf. arrow in Fig. 1B). Inhibition of the Ca2+ by La3+ was analysed in the same manner. Fluorescence-quench experiments were monitored in the presence of 500 µM MnCl2 at an excitation wavelength of 359 nm (corresponding to the isosbestic point) and an emission wavelength of 510 nm.

Application of compounds to single cells was performed by a superfusion system with a 7-channel perfusion pipette (List-electronic, Darmstadt, Germany), driven by a valvebank (TSE, Bad Homburg, Germany) with solution exchange times of less than 500 ms. For experiments we used multinucleated cells only which showed a rise in [Ca2+]i when challenged with Ca2+ free depolarisation solution (depolarisation solution HK: Tyrode's solution, but with 60 mM KCl and 80 mM NaCl). Based on the current model of skeletal muscle EC coupling, these cells fulfil essential criteria for the presence of intact functional coupling between the L-type Ca2+ channel and the RyR type 1. During measurements cells were constantly rinsed with fresh Tyrode's solution (concentrations in mM: NaCl 137, glucose 5.6, KCl 5.4, NaHCO3 2.2, MgCl2 1.1, NaH2PO4, HEPES/Na 10, CaCl2 1.8, pH 7.4). Ca2+ free assay conditions were obtained by omitting Ca2+ and addition of 0.5 mM EGTA in Tyrode's solution. Application of compounds in the absence of Ca2+ was preceded for a few seconds by a short rinse of the cells with Ca2+ free solution to exclude an effect of Ca2+ deprivation.

In addition, fluorescence photometry was carried out with muscle cells in suspension using a Hitachi F-4500 photometer. Differentiated myotubes (2–15×106 cells) were loaded with 4–10 µM fura2/AM (supplemented with 0.025% pluronic acid) for 35 and 60 min at 37°C in Tyrode's solution. The cells were washed twice and finally resuspended in Tyrode's solution. Mn2+ influx was monitored at an excitation wavelength of 359 nm (corresponding to the isosbestic point evaluated at the photometer) and an emission wavelength of 510 nm in a manner analogous to the Ca2+ imaging experiments. Similar to single cell Ca2+ imaging differentiated human skeletal muscle cells in suspension responded to depolarisation with HK in the absence of extracellular Ca2+, caffeine or nifedipine. Undifferentiated skeletal muscle cells did not show an elevation in [Ca2+]i by the application of nifedipine which was in the line of our previous findings in single cell Ca2+ imaging (Weigl et al. 2000). Light sensitive compounds were kept in dark until use and exposed to light for measurements not longer than 2 min.

RT-PCR experiments

Total RNA was isolated from differentiated human skeletal muscle cells or rat hippocampal neurones using the RNeasy Mini-kit from Qiagen. Subsequently, total RNA was reverse transcribed into first-strand cDNA with the RT-PCR-kit from Takara using oligo-dT and random primers. Aliquots of the cDNA were employed as templates for PCR amplification with specific primers for human TRPC 1, 3 and 4 taken from Groschner et al. (1998) and for TRPC 5 and 6 from Garcia and Schilling (1997). In control experiments, PCR was carried out with the following templates: total RNA, total RNA treated with DNase I or in the absence of a template. All these experimental conditions did not result in the amplification of positive PCR products. As a positive control TRPC 5 primers were used under identical PCR conditions with rat hippocampal cDNA as a template leading to the amplification of a 340 bp PCR product of the predicted size. The products were resolved on a 1.5% agarose gel by electrophoresis. In addition the PCR products were sequenced and found to be identical to the respective human isoforms.

Miscellaneous procedures

All experiments were repeated at least three times. Data are given as mean ± SEM unless otherwise stated. Statistical analysis for multiple comparison was performed using ANOVA and a subsequent post hoc Scheffe's test. For single comparison Student's t-test was carried out to test the significance of differences; a value of p<0.05 was considered statistically significant.

Results

Store-operated Ca2+ influx in human skeletal muscle cells

Differentiated primary human skeletal muscle cells typically responded with a Ca2+ transient in the absence of extracellular Ca2+ when depolarised by high potassium concentrations (HK) (Fig. 1A). The formation of an intact triad, namely the apposition of the voltage-sensitive L-type Ca2+ channels in the plasma membrane and the RyR type 1 of the sarcoplasmic reticulum, is a prerequisite for such HK induced intracellular Ca2+ transients (Franzini-Armstrong 2000; Schneider 1994). In contrast, simple omission of extracellular Ca2+ in the bath solution had no effect on the fura2 fluorescence trace (Fig. 1A). Omission and re-addition of extracellular Ca2+ was not sufficient to trigger a Ca2+ release or Ca2+ influx (Fig. 1A). Caffeine which directly activates the RyR type 1, triggered intracellular Ca2+ transients although extracellular Ca2+ was not present (Fig. 1B) (Herrmann-Frank et al. 1999; Hohenegger et al. 1996; Klinger et al. 1999; Liu and Meissner 1997). Typically, after application of caffeine in the absence of extracellular Ca2+, an increase in the [Ca2+]i trace was detectable upon Ca2+ re-addition to the extracellular bath solution (indicated by an arrow in Fig. 1B). We compared the basal Ca2+ level in the absence of extracellular Ca2+ with the peak Ca2+ level in the presence of extracellular Ca2+ 15 s after the end of a 30 mM caffeine pulse. The intracellular Ca2+ concentration was augmented from 62.8±4.5 nM to 101.6±11.4 nM (p<0.005; n=23). This elevation in the Ca2+ signal could be completely suppressed by La3+ which was previously found to block store operated Ca2+ influx (Kwan et al. 1990; Okada et al. 1998; Robertson et al. 2000) (cf. Fig. 1B, D). The caffeine induced Ca2+ transient is strictly dependent on filled Ca2+ stores. Depletion of intracellular Ca2+ stores by thapsigargin, a blocker of the Ca2+ ATPase of the sarcoplasmic reticulum (Thastrup et al. 1994) completely prevented caffeine induced Ca2+ responses (Fig. 1C).

Fig. 1A–D.
figure 1

Voltage-insensitive Ca2+ influx is present in human skeletal muscle cells. Differentiated human skeletal muscle cells loaded with fura2/AM were kept in Tyrode's solution for Ca2+ fluorescence imaging (grey bars; Ca2+=1.8 mM) or challenged in the absence of Ca2+ (Tyrode's solution without Ca2+ but with 0.5 mM EGTA; white bars; Ca2+=0). Multinucleated cells which responded to depolarisation solution (HK) in the absence of extracellular Ca2+ were considered to be differentiated skeletal muscle cells. A Switching from Ca2+ containing medium to Ca2+ free medium and back to Ca2+ containing extracellular solution did not alter intracellular Ca2+ concentrations. B [Ca2+]i was monitored upon application of depolarisation solution (HK), 30 mM caffeine and 1 mM La3+. Caffeine-induced Ca2+-release was observed in the absence of extracellular Ca2+ (white bars) and was followed by a Ca2+ influx (arrow). Subsequent Ca2+ influx upon extracellular Ca2+ re-addition (grey bars) is inhibited by La3+. C After depolarisation of a skeletal muscle cell with HK in the absence of extracellular Ca2+ the cells were treated with 5 µM thapsigargin for the indicated time and subsequently exposed to 30 mM caffeine and 1 mM LaCl3 as indicated. D After the addition of 30 mM caffeine in the absence of extracellular Ca2+ subsequent Ca2+ influx (as indicated in panel B by an arrow) was quantified. The increment in intracellular Ca2+ concentration was measured 15 s after ceasing caffeine application and expressed as Δ[Ca2+]i. Ca2+ influx was completely suppressed by 1 mM La3+ (Student's t-test, p<0.0005). The bars indicate the mean and SD; the number of experiments (n) is given in parenthesis

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As already seen in Fig. 1B (indicated by an arrow), the tail of the caffeine induced Ca2+ transient was elevated upon switching from an extracellular solution free of Ca2+ (white bars) to a Ca2+ containing medium (grey bars). In thapsigargin treated cells this buffer change evoked the same phenomenon of Ca2+ influx (Fig. 1C), but was independent of the presence of caffeine. Again this store operated Ca2+ influx was completely blocked by 1 mM La3+ in a reversible manner and was only seen when Ca2+ was supplemented in the extracellular bath solution (grey bars in Fig. 1C). As depicted in Fig. 1, store operated Ca2+ influx was inhibited by La3+, irrespective of whether store depletion was induced by caffeine (Fig. 1B, D) or thapsigargin (Fig. 1C). In both cases, upon wash-out of La3+ the inhibition was reversed.

It is yet not clear from the literature which voltage-insensitive Ca2+ influx channels are present in human skeletal muscle. Assuming that TRPC channels participate in Ca2+ influx it should be possible to detect their message using RT-PCR. On transcriptional level human TRPC1, TRPC3, TRPC4 and TRPC6 channels are present in human skeletal muscle cells (Fig. 2). Using a temperature gradient during the PCR reaction we controlled simultaneously for the specificity of the PCR products. At a very low primer annealing temperature of 42°C a non-specific band is visualised for TRPC 3 isoform which was vanished at higher temperatures. Nevertheless, all PCR products were purified from agarose gels and their identity was confirmed by sequencing. The primer pairs were chosen in order to obtain products that crossed intron-exon borders. This implies that the mRNA for TRPC 1, 3, 4 and 6 channels is correctly transcribed and spliced, and the observed products did not result from a contamination of genomic DNA.

Fig. 2.
figure 2

RT-PCR products for specific human TRPC channel isoforms. PCR products from RT-PCR with specific primers for the given isoforms of human TRPC channels were resolved on a 1.5% agarose gel and visualised by ethidium bromide. The expected size of PCR products are given in parenthesis and the different annealing temperatures indicated below. The melting temperatures calculated from the forward and reverse primer sequence were 50°C and 53°C for TRPC 1, 48°C and 44°C for TRPC 3, 60°C and 54°C for TRPC 4, 48°C and 58°C for TRPC 5 and 48°C and 59°C for TRPC 6, respectively. The molecular mass of the 100 bp-DNA ladder standard (std.) is indicated by the arrows

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Linkage of the ryanodine-sensitive Ca2+ pool to Ca2+ influx

In order to further confirm a link between the ryanodine-sensitive Ca2+ pool and store operated Ca2+ influx, additional experiments were carried out using the dihydropyridine, nifedipine. We recently demonstrated, that the L-type Ca2+ channel antagonist nifedipine is able to induce Ca2+ release from ryanodine-sensitive Ca2+ pools in human skeletal muscle cells, although under these conditions the L-type Ca2+ channel is inhibited and no Ca2+ influx can occur via this channel (Weigl et al. 2000). Therefore nifedipine was suitable as a second compound to selectively release Ca2+ from ryanodine-sensitive Ca2+ pools but now under conditions where we can exclude Ca2+ influx via the L-type Ca2+ channel (dihydropyridine receptor). Again, when intracellular Ca2+ stores in human skeletal muscle cells were depleted with 1 µM thapsigargin, similar to the conditions given in Fig. 1C, the basal intracellular Ca2+ concentration was elevated. Under such conditions the simple omission of extracellular Ca2+ (Fig. 3, white bars) followed by re-addition of Ca2+ (Fig. 3, grey bars) led to a Ca2+ influx. Nifedipine was not able to induce a Ca2+ transient in thapsigargin treated cells; independent whether extracellular Ca2+ was absent or present (Fig. 3).

Fig. 3A, B.
figure 3

Nifedipine induced intracellular Ca2+ transients and Ca2+ influx. Fura2/AM loaded differentiated human skeletal muscle cells which reacted to HK with a [Ca2+]i transient (not shown) were incubated in 1 µM thapsigargin for 15 min. A Sequentially, the cells were exposed to Ca2+ containing Tyrode's solution (grey bars) and Ca2+ free Tyrode's solution (white bar). Under conditions of depleted intracellular Ca2+ stores nifedipine was not able to trigger a Ca2+ transient in the presence of extracellular Ca2+. B Under the same conditions of thapsigargin treatment myotubes were subjected to 10 µM nifedipine (Nif) either in the presence or absence of extracellular Ca2+. Under conditions of depleted Ca2+ stores, the change in [Ca2+]i is solely dependent on the extracellular [Ca2+] and not on the presence of nifedipine

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Conversely, in differentiated muscle cells which were not exposed to thapsigargin, 10 µM nifedipine clearly evoked a Ca2+ transient (Fig. 4B). The Ca2+ transient induced by nifedipine was broader compared to caffeine and seems to be composed of a second sustained Ca2+ release phase (cf. Fig. 1B with Fig. 4B). In the presence of extracellular Ca2+ the transient lasted for the duration of the drug application and was fully reversible upon wash-out (Fig. 4B). This is consistent with our previous findings (Weigl et al. 2000). Simultaneous addition of 100 µM Cd2+, an inhibitor of voltage sensitive and insensitive Ca2+ channels (Parekh and Penner 1997), did not prevent the initiation of the nifedipine induced Ca2+ transient. However, it turned the sustained shape of the nifedipine induced Ca2+ elevation into a sharp Ca2+ transient (Fig. 4A). We have quantified this effect in the absence and presence of Cd2+ by comparing the peak amplitude of the nifedipine induced Ca2+ transient with the Ca2+ concentration after 50 s of drug application. The intracellular Ca2+ concentration at this time point was 92.3±3.0% (n=5) of the peak amplitude in the absence of Cd2+. In the presence of 100 µM Cd2+ the intracellular Ca2+ concentration was reduced to 43.9±7.0% (n=5; p=0.0007) of the peak amplitude of the nifedipine induced Ca2+ transient. This confirms our previous finding that binding of nifedipine to the dihydropyridine binding site of the L-type Ca2+ channel is sufficient to trigger the RyR type 1 opening (Weigl et al. 2000). Ca2+ influx initiated by the application of 10 mM caffeine was also inhibited by 100 µM Cd2+, while the caffeine induced Ca2+ release was unaffected (data not shown).

Fig. 4A–C.
figure 4

Nifedipine induced Ca2+ influx is inhibited by Cd2+ and La3+. A Nifedipine induced Ca2+ release in fura2 loaded differentiated skeletal muscle cells was independent of whether 100 µM CdCl2 was present prior to the nifedipine addition or added afterwards. The arrows indicate a Ca2+ elevation. B Similar to panel A. A differentiated skeletal muscle cell loaded with fura2 was repeatedly exposed to 10 µM nifedipine (Nif). The cell was sequentially exposed to Ca2+ containing Tyrode's solution (grey bars; 1.8 mM Ca2+) or Ca2+ free Tyrode's solution (white bars; zero Ca2+). The addition of 300 µM La3+ blocked the Ca2+ influx component of the nifedipine-induced Ca2+ transient similar to Cd2+. C The Ca2+ influx induced by 10 µM nifedipine was quantified similar to Fig. 1D. The Ca2+ influx was significantly inhibited in the presence of 300 µM La3+ (Student's t-test, p=0.01). The bars indicate the mean and SD; the number of experiments (n) is given in parenthesis

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Similar to caffeine, nifedipine significantly triggered a Ca2+ influx (control: 83.8±5.0 nM; peak [Ca2+]i after 10 µM nifedipine 107.0±6.8 nM; p=0.01; n=12) (Fig. 4B, C). Again lanthanum, which inhibited caffeine induced Ca2+ influx (Fig. 1D), significantly suppressed nifedipine induced Ca2+ influx (Fig. 4B, C). The sensitivity of the Ca2+ influx to Cd2+ and La3+ provided evidence for participation of TRPC channels on this phenomenon (Parekh and Penner 1997).

Visualisation of Ca2+ influx by Mn2+ induced quenching of fluorescence

A common method to visualise Ca2+ influx is to make use of the isosbestic wavelength of fura2. Upon excitation at 359 nm, fura2 does not respond to changes in [Ca2+]i. However, a Mn2+ influx through plasmalemmal Ca2+ channels quenches the intracellular fluorescence signal which is therefore proportional to the Mn2+ influx (Hopf et al. 1996a; Kass et al. 1994; Kurebayashi and Ogawa 2001; Xu et al. 1994). Using the Ca2+ imaging technique, simultaneous recordings of the fluorescence signal of fura2 loaded differentiated skeletal muscle cells excited at 340 nm, 359 nm and 380 nm are presented in Fig. 5. Usually, in the presence of HK or caffeine the muscle cells contracted which caused an artefact in the 359 nm fluorescence trace. However, upon addition of 500 µM Mn2+, a basal Mn2+ influx was observed which was clearly accelerated by the co-application of 10 mM caffeine or 10 µM nifedipine (Fig. 5A, B, respectively). The slopes were calculated by linear regressions and used as estimates of the Mn2+ influx rate. The basal rate of Mn2+ influx (0.6±0.06 a.u./s; mean ± SEM, n=8) was significantly increased two- (1.05±0.13 a.u./s; mean ± SEM, p<0.05; n=3) and three-fold (1.94±0.31 a.u./s; mean ± SEM, p<0.005; n=4) in the presence of nifedipine and caffeine, respectively.

Fig. 5A, B.
figure 5

Caffeine and nifedipine trigger Mn2+ influx in skeletal muscle cells. A Fluorescence of a fura2 loaded myotube was monitored simultaneously at an excitation wavelength of 340 nm and 380 nm (thick line) and at its isosbestic point (excitation wavelength 359 nm; dotted line). Horizontal lines indicate the drug application. Basal Mn2+ influx was triggered by the addition of 10 mM caffeine (Caff.) and resulted in an acceleration of fluorescence quenching. The regression lines of the isosbestic fluorescence trace before and after caffeine addition are given as overlays. B Under conditions similar to panel A Mn2+ influx was also stimulated by 10 µM nifedipine (Nif). The Ca2+ content of the extracellular solution is given by white (zero Ca2+) and grey (1.8 mM Ca2+) bars

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Ca2+ influx in skeletal muscle cell suspensions

The results for the fura2 loaded skeletal muscle cells in cell suspension were similar compared to the results presented on single cell level in Fig. 5. Mn2+ was added to the extracellular bath solution which resulted in a rapid drop in fluorescence intensity correlating to the quenching of extracellular fura2 (Fig. 6A). This initial step in fluorescence was followed by a basal Mn2+ influx comparable to the isosbestic fluorescence traces in Fig. 5. SKF96365, an inhibitor of voltage-insensitive Ca2+ influx (Okada et al. 1998; Zhu et al. 1998), per se had no effect on the isosbestic fluorescence (insert in Fig. 6A; Ex: 359 nm, Em: 510 nm). However, SKF96365 inhibited Mn2+ influx immediately (Fig. 6A). The fluorescence traces were again analysed by linear regressions and expressed as arbitrary fluorescence units (a.u.) per time and normalised to the basal Mn2+ influx in the absence of any drug. The initial quenching of free fura2 was not introduced into the calculation. Analogous to experiments on single cell level, basal Mn2+ influx was further enhanced by intracellular Ca2+ store depletion due to application of nifedipine or caffeine (Fig. 6B). SKF96365 blocked the Mn2+ influx irrespective of either nifedipine or caffeine was used to elicit it. Although SKF96365, La3+ and Cd2+ have been described to be not specific for store operated Ca2+ influx channels they have been extensively used and shown to block some TRPC channels (Parekh and Penner 1997). In conclusion of our observations, the Ca2+ influx and Mn2+ influx was abolished by SKF96365, La3+ and Cd2+ in human skeletal muscle cells, not only on single cell level but also in mass suspension.

Fig. 6A, B.
figure 6

SKF96365 inhibits Mn2+ influx in human skeletal muscle cells. A Fura2 loaded differentiated human skeletal muscle cells (2–4×105 cells) were kept in Tyrode's solution and used for fluorescence photometry at the isosbestic point of fura2 to monitor the fluorescence quenching by Mn2+. The application of 500 µM MnCl2 and 30 µM SKF96365 is indicated by the arrows. 30 µM SKF96365 per se had no effect on the fluorescence signal as given in the inset of panel A. B The percentage of the activation and inhibition of the Mn2+ influx was employed under conditions similar to panel A. The Mn2+ influx was stimulated by 10 mM caffeine (Caff; n=12), 10 µM nifedipine (Nif; n=9) and inhibited by 30 µM SKF96365 in the absence (SKF; n=10) or presence of 10 mM caffeine (SKF/Caff; n=7) and 10 µM nifedipine (SKF/Nif; n=10). Data in panel B were normalised by setting the basal Mn2+ influx in the absence of any drug 100%. The mean percentage of the Mn2+ influx is depicted and the standard deviation indicated by error bars. Single factor analysis of variance (ANOVA) of the data and subsequent Scheffe's post hoc comparison resulted in significance for all drug applications considering a p value <0.05

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Ca2+ influx by the depletion of ryanodine-sensitive Ca2+ pools is independent of phospholipase C activity

Hitherto, it is clear that depletion of the ryanodine-sensitive Ca2+ pool suffices to trigger Ca2+ influx from the extracellular compartment. 1,2-Dioctanoyl-sn-glycerol (DOG), a cell permeable analogue of diacylglycerol should activate TRPC3 and TRPC6 channels (Hofmann et al. 1999), which were present on mRNA level in skeletal muscle cells (Fig. 2). DOG was dissolved in DMSO which did not exceed a final concentration of 0.1% (v/v), a concentration which was without effects on [Ca2+]i (Weigl et al. 2000). Furthermore, it should be expected that an inhibitor of the diacylglycerol lipase, RHC-80267, further amplifies this Mn2+ influx. In fact, basal Mn2+ influx in the presence of 100 µM DOG was increased to 120.1±4.4% (n=7; mean ± SD) (Fig. 7). When 80 µM RHC-80267 was applied and successively followed by the addition of 100 µM DOG the increment in Mn2+ influx reached 144.4±15% (n=4; mean ± SD). By comparison, Mn2+ influx evoked by 10 mM caffeine or 10 µM nifedipine was not altered by the co-application of the diacylglycerol lipase inhibitor (Fig. 7B). The sole addition of 80 µM RHC-80267 had no detectable effect on Mn2+ influx.

Fig. 7A, B.
figure 7

Diacylglycerol does not participate in caffeine or nifedipine stimulated Mn2+ influx. A Under conditions given in the legend of Fig. 6A the fluorescence signal of fura2 at the isosbestic point was determined after the application of 500 µM MnCl2. The effect on basal Mn2+ influx (set 100%; open bar, n=11) was compared to the changes in steepness of the Mn2+ influx due to subsequent addition of 100 µM DOG (DOG; hatched bar, n=7) and 100 µM DOG with 80 µM RHC-80267 (DOG/RHC; cross hatched bar, n=4). B Under identical conditions as in panel A the effect of RHC-80267 on Mn2+ influx was investigated. Basal Mn2+ influx was set 100% (open bar; n=15) and stimulated either by 10 mM caffeine (Caff; n=4), 10 µM nifedipine (Nif; n=4) or 80 µM RHC-80267 (RHC; n=4). Simultaneously, 500 µM MnCl2 plus 80 µM RHC-80267 was applied and with a delay of 2 min 10 mM caffeine (Caff/RHC; n=5) or 10 µM nifedipine (Nif/RHC; n=5) were added. The bars indicate the mean percentage of the Mn2+ influx determined by linear regression and the standard error of mean is indicated by error bars

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The DOG induced acceleration in Mn2+ influx may be due to an activation of a protein kinase C. Therefore, Mn2+ influx was investigated in the absence and presence of inhibitors of the protein kinase C. GF109203X and staurosporine, two protein kinase C inhibitors, had no influence on basal Mn2+ influx (Table 1). The addition of DOG or the combination of DOG plus RHC-80267 augmented basal Mn2+ influx significantly. The same was true for caffeine and nifedipine. Interestingly, the protein kinase C inhibitors had no effect on the increment of DOG induced Mn2+ influx. In contrast, the Mn2+ influx evoked by DOG plus RHC-80267 was significantly inhibited by 65%. This finding may support the conjecture that a protein kinase C is involved in DOG plus RHC-80267 mediated Mn2+ influx. Data for Mn2+ influx triggered by caffeine or nifedipine were controversial in the presence of protein kinase C inhibitors. GF109203X inhibited caffeine triggered Mn2+ influx slightly (17%). However, in all other cases protein kinase C inhibitors did not alter caffeine or nifedipine induced Mn2+ influx. In staurosporine pre-treated cells, nifedipine induced Mn2+ influx was significantly augmented (44% stimulation; p<0.05). Overall, the acceleration in Mn2+ influx was much more pronounced when skeletal muscle cells were allowed to differentiate for 10 days (Table 1) compared to 4 days in experiments shown in Figs. 6 and 7. Taken together, the data presented so far demonstrate that caffeine and nifedipine accelerate Mn2+ influx independently of the presence of the second messenger diacylglycerol (Fig. 7). While DOG plus RHC-80267 induced Mn2+ influx is completely blocked by protein kinase C inhibitors, the participation of protein kinase C in caffeine or nifedipine induced Mn2+ influx is unlikely (Fig. 7, Table 1).

Table 1. Diacylglycerol dependent Mn2+ influx but not caffeine or nifedipine induced Mn2+ influx is dependent on protein kinase C. Fura2 loaded differentiated skeletal muscle cells (5–8×105) were exposed to Tyrode's solution in the absence (control) or presence of the protein kinase C inhibitors GF109203X or staurosporine. After 2 min of incubation with the PKC inhibitors 500 µM MnCl2 (basal Mn2+ influx) or 500 µM MnCl2 plus 100 µM DOG, 500 µM MnCl2 plus 100 µM DOG and 80 µM RHC-80267 (RHC), 500 µM MnCl2 plus 10 mM caffeine or 500 µM MnCl2 plus 10 µM nifedipine was added. Asterisks and crosses indicate level of significance obtained from multiple comparison analysis with ANOVA and post hoc Scheffe's test
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Although improbable, formally it cannot be ruled out that caffeine or nifedipine induced Mn2+ influx is dependent on the activation of the InsP3 receptor. We have used the phospholipase C inhibitor U73122 to block the synthesis of InsP3 (Berridge et al. 1998). Concentrations of 5–25 µM U73122 were not able to inhibit Mn2+ influx triggered by caffeine or nifedipine (data not shown). As already mentioned transactivation of the InsP3 receptor by elevated [Ca2+]i represents another possibility to explain Ca2+ influx after depletion of ryanodine sensitive Ca2+ stores. In order to address this question, we have exposed differentiated muscle cells to the cell permeable InsP3 receptor inhibitor xestospongin C. Surprisingly, the resting Ca2+ concentration under xestospongin C treatment was augmented from 59.5±13.8 a.u. (n=17) to 216±25.6 a.u. (mean ± SD; n=21; p<0.001). This observation may be due to unspecific effects of xestospongin C (Bootmann et al. 2002). In particular, inhibition of the Ca2+ ATPase of the sarcoplasmic reticulum is a likely explanation for the increase in [Ca2+]i as this is observed also for other Ca2+ ATPase inhibitors like thapsigargin (De Smet et al. 1999). Nevertheless, in the presence of 10 µM xestospongin C, caffeine or nifedipine induced Mn2+ influx was not altered in differentiated human skeletal muscle cells (data not shown). In conclusion, neither the phospholipase C products InsP3 and diacylglycerol nor protein kinase C contributed to a Mn2+ influx induced by selective depletion of ryanodine-sensitive Ca2+ pools by caffeine or nifedipine.

Discussion

In the present study we investigated voltage-insensitive Ca2+ influx by selective depletion of the ryanodine-sensitive Ca2+ pool in differentiated human skeletal muscle cells. The mechanism of activation of this Ca2+ influx did not involve phospholipase C, protein kinase C or the InsP3 receptor. These conclusions are based on the following observations:

  1. 1.

    Ca2+ influx occurs if the intracellular Ca2+ release is evoked by caffeine. While unlikely, it is not possible to formally exclude Ca2+ influx through L type Ca2+ channels (Figs. 1, 5)

  2. 2.

    We therefore used nifedipine to release Ca2+ via the RyR type 1. Although the L type Ca2+ channel is blocked under these conditions (Weigl et al. 2000), nifedipine induced Ca2+ influx (Figs. 4, 5)

  3. 3.

    Neither caffeine nor nifedipine were able to induce Ca2+ transients in thapsigargin treated skeletal muscle cells, which highlights the fact that both compounds trigger Ca2+ release and that this stimulus is the signal for Ca2+ influx (Figs. 1, 3)

  4. 4.

    Irrespective of whether Ca2+ influx was provoked by caffeine or nifedipine, it was not altered under conditions where the phospholipase C, protein kinase C or InsP3 receptor were inhibited. Nevertheless, in staurosporine treated cells nifedipine induced Mn2+ influx was significantly enhanced (Table 1). This observation is unclear at the moment

  5. 5.

    Furthermore, the application of the diacylglycerol lipase inhibitor, RHC-80267, did not have an effect on caffeine or nifedipine induced Ca2+ influx (Fig. 7). These data allow the assumption that the observed Ca2+ influx due to nifedipine or caffeine is independent of products of the phospholipase C pathway, InsP3 and diacylglycerol

In skeletal muscle for the first time Ca2+ influx through Ca2+ leak channels was shown by Hopf et al. (1996a). In mouse myotubes Ca2+ influx was provoked by the reversible Ca2+ ATPase inhibitor, cyclopiazonic acid. Similar to our experiments this Ca2+ influx was visualised by Mn2+ quench experiments and sensitive to La3+. We also found store operated Ca2+ influx when the Ca2+ ATPase of the sarcoplasmic reticulum was blocked by thapsigargin (Figs. 1C, 3). This mechanism was also conserved in isolated skeletal muscle fibres where Kurebayashi and Ogawa (2001) detected Mn2+ influx when Ca2+ stores were depleted by Ca2+ ATPase inhibitors.

Recently, we found that the dihydropyridines nifedipine and nitrendipine increase the intracellular Ca2+ concentration with an EC50 of about 0.6 and 1.2 µM, respectively. This rise in [Ca2+]i is due to activation of the RyR type 1 and subsequent entry of Ca2+ (Weigl et al. 2000). Hopf et al. (1996b) also found activation of Ca2+ leak channels by high concentrations of nifedipine (50 µM) in mouse myotubes and attributed this effect to direct activation of leak channels by this dihydropyridine. However, we previously showed that this rise in [Ca2+]i is dependent on the intact EC-coupling mechanism and can be prevented e.g. by depolarisation of the cells to −30 mV which involves inactivation of the DHP receptor. In depolarised skeletal muscle cells nifedipine never induces Ca2+ release or Ca2+ influx (Weigl et al. 2000). Therefore we conclude that the Ca2+ influx triggered by nifedipine depends on intact EC-coupling and is not due to a direct activation of leak channels as proposed by Hopf at al. (1996b). The fact that Ca2+ influx occurs in the presence of 10 µM nifedipine which already blocks DHP receptors at nanomolar concentrations implies that this Ca2+ influx can not occur via DHP receptors and shows that there exists a mechanism which is able to replenish emptied ryanodine sensitive Ca2+ stores independently of the DHP receptor opening.

Candidates for ion channels which mediate voltage independent Ca2+ influx are the TRPC channels. Correctly spliced RT-PCR products were found for TRPC1, TRPC3, TRPC4 and TRPC6 channels in human myotubes (Fig. 2). The type 5 isoform of TRPC channels is expressed exclusively in the brain and was positive in the hippocampus (data not shown); accordingly, in myotubes we did not detect the mRNA of TRPC5 channel (Fig. 2; Philipp et al. 1998; Okada et al. 1998). Depending on the cell system TRPC3 and TRPC6 channels were either proposed to be store operated Ca2+ channels (Boulay et al. 1997; Mizuno et al. 1999) or activated via PLC linked receptors (Hofmann et al. 1999; Zitt et al. 1997). The direct activation of TRPC3 and TRPC6 channels by diacylglycerol was first described by Hofmann and co-workers (Hofmann et al. 1999). In line with these findings we also observed stimulation of Mn2+ influx by the cell permeable diacylglycerol analogue, DOG (Fig. 7A, Table 1). DOG was apparently metabolised rather rapidly, because co-application of the diacylglycerol lipase inhibitor, RHC-80267, clearly further accelerated the Mn2+ influx in skeletal muscle cells (Fig. 7A, Table 1). Interestingly, the protein kinase C inhibitors, GF109203X and staurosporine, completely suppressed DOG plus RHC-80267 induced Mn2+ influx. In contrast, we failed to elucidate a participation of protein kinase C on DOG induced Mn2+ influx. Possibly, inter-assay variability of the preparation of human skeletal muscle cells and the small amplitude of the DOG induced increment in Mn2+ influx may explain this observation. However, when caffeine or nifedipine were administrated simultaneously with RHC-80267 the Mn2+ influx was not altered, indicating that protein kinase C is not involved in this Mn2+ influx. It is worth to mention that a member of the superfamily of TRP channels, the TRPM7 channel has been identified to contain a huge phospholipase C-interacting kinase at the COOH-terminal end which is functionally linked to conductivity of the ion channel (Runnels et al. 2001).

In conclusion, these data show that in skeletal muscle at least two independent Ca2+ influx mechanisms exist which can be separated pharmacologically. The depletion of ryanodine-sensitive Ca2+ stores is coupled to Ca2+ influx independent of phospholipase C, protein kinase C and the InsP3 receptor. However, a phospholipase C dependent and protein kinase C dependent Ca2+ influx pathway obviously exists in human myotubes but is not involved in Ca2+ influx due to emptying of ryanodine-sensitive Ca2+ stores. We favour a model where the emptying of ryanodine-sensitive Ca2+ stores is coupled more directly to Ca2+ influx. The observation by Kiselyov et al. (2000) provides biochemical and functional evidence that ion channel tandems (TRPC3 channel/InsP3 receptor and TRPC3 channel/ryanodine receptor) cluster and segregate in plasma membrane microdomains. Co-localisation of these ion channel complexes in the skeletal muscle can account for a signalling cascade that is triggered in parallel to EC-coupling. In a recent publication the synaptophysin-family-related protein, mitsugumin 29 was attributed to guide the ryanodine receptor to store operated Ca2+ channels (Pan et al. 2002). Store operated Ca2+ influx was inhibited by SKF96365> 90% and strictly dependent on the presence of the ryanodine receptor type 1 which is consistent with our findings (Fig. 6). In myotubes deficient in mitsugumin 29, store operated Ca2+ influx was virtually absent (Pan et al. 2002).

Voltage-independent Ca2+ influx plays a crucial role in the amplification, prolongation and modification of intracellular Ca2+ transients for example in T-lymphocytes (Alberola et al. 1997; Berridge 1997; Guse 1998). It is attractive to speculate that the implications of analogous mechanisms operate in skeletal muscle EC-coupling and are of potential relevance in the physiology and pathophysiology of skeletal muscle (Hopf et al. 1996b).