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Differential regulation of calcium channel coding genes by prolonged depolarization
Neurochemistry International 50 (2007) 395–403 www.elsevier.com/locate/neuint
Differential regulation of calcium channel coding genes by prolonged depolarization A. Benavides, D. Pastor, N. Fradejas, D. Tornero, S. Calvo * Faculty of Medicine and Regional Center for Biomedical Research, University of Castilla La Mancha, Albacete, Spain Received 18 July 2006; received in revised form 14 September 2006; accepted 18 September 2006 Available online 23 October 2006
Abstract Calcium channels must be subjected to a very precise regulation in order to preserve cell function and viability. Voltage gated calcium channels (VGCC) represent the main pathway for calcium entry in excitable cells. This explains why depolarization induces a rapid-onset and short-term inactivation of calcium currents. Contrarily to this well-documented mechanism to maintain calcium below toxic levels, the regulatory pathways inducing longer-lasting changes and cell surface expression of functional calcium channels are largely unknown. Since calcium is a main player in the activity-dependent regulation of many genes, we hypothesize that calcium channel coding genes could be also subjected to activity-dependent regulation. We have used prolonged depolarization to analyze the effects of sustained intracellular calcium elevation on the mRNAs coding for the different a1 pore-forming subunits of the calcium channels expressed in chromaffin cells. Our findings reveal that persistent depolarization is accompanied by a prolonged intracellular calcium elevation and reduction of calcium current. This calcium current inhibition could be mediated, at least partially, by the downregulation of the mRNAs coding for several a1 subunits. Thus, we show here that depolarization inhibits the expression of CaV1.1, CaV1.2, CaV1.3, CaV2.2 and CaV2.3 mRNAs, while the CaV2.1 mRNA remains unmodified. Moreover, such downregulation of channels depends on calcium entry through the L-type calcium channel, as both mRNA and calcium current changes induced by depolarization are abrogated by L-type channel specific blockers. # 2006 Elsevier Ltd. All rights reserved. Keywords: Calcium channel expression; CaV; Transcription; Activity-dependent gene regulation
1. Introduction Voltage-gated Ca2+ channels (VGCC) are of great importance in coupling excitability to many Ca2+-dependent events within the cells. The Ca2+ channels are complex proteins composed of several (a1, a2d, b and g) subunits. The a1 subunit forms the ionconducting pore and contains the voltage sensor and the interaction sites for channel blockers and activators (Hofmann et al., 1999). The auxiliary subunits a2d, b and g modulate channel properties such as inactivation and channel targeting to the membrane (Klugbauer et al., 1999; Perez-Garcia et al., 1995). Bovine adrenal chromaffin cells express at least four types of voltage-gated Ca2+ channels: L, N, P/Q and R (Aldea et al., 2002) encoded by, at least, six different a1-subunit genes:
* Corresponding author at: Centro Regional de Investigaciones Biome´dicas, Universidad de Castilla La Mancha, Avda. de Almansa 14, 02006 Albacete, Spain. Tel.: +34 967599200x2956; fax: +34 967599324. E-mail address: [email protected] (S. Calvo). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.09.013
CaV1.1, CaV1.2 and CaV1.3 for L-type, CaV2.1, CaV2.2 and CaV2.3 for P/Q-, N- and R-types, respectively (Benavides et al., 2004). These channels coexist in single cells and represent the main physiological trigger for catecholamine release (Aldea et al., 2002). The intracellular Ca2+ concentration must be kept below certain limits to allow cell functionality. It is well known that elevated intracellular Ca2+ concentrations are toxic for the cells and induce cell death (for review, see Orrenius et al., 2003). Moreover, catecholamine secretion by adrenal medulla chromaffin cells must be also strictly regulated to avoid excessive release of catecholamines which could cause irreversible effects such as heart failure, pulmonary edema or hypertension-linked comas (Aunis and Langley, 1999). Ca2+ influx through voltage-activated Ca2+ channels in chromaffin cells must be, therefore, finely tuned. Mammalian cells have developed different mechanisms to avoid maintained or excessive Ca2+ increases. Among them, Ca2+ binding proteins, intracellular reservoirs and extrusion pumps are the most significant. Additionally, VGCC inactivation
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by both, voltage and Ca2+ itself, in a short-term, membranedelimited fashion, also contributes to avoid intracellular Ca2+ overload (reviewed in Budde et al., 2002; Stotz and Zamponi, 2001). There is also compiling evidence pointing at the existence of a longer-lasting Ca2+ channel regulation induced by prolonged depolarization and sustained Ca2+ increases. In this sense, high potassium-induced downregulation of L-type Ca2+ currents has so far been described in myenteric neurons (Franklin et al., 1992); clonal pituitary cells GH4C1 (Liu et al., 1994; Peri et al., 2001); PC12 cells (DeLorme et al., 1988; Feron and Godfraind, 1995); frog skeletal muscle fibers (Carrillo et al., 2004; Escamilla et al., 2001); chick retina and rat cardiac cells (Ferrante et al., 1991). Ca2+ currents have also been reported to decrease in response to chronic electrical stimulation in dorsal root ganglion neurons (Li et al., 1996) and crayfish motoneurons (Hong and Lnenicka, 1995; Lnenicka and Hong, 1997). Opposite effects have been observed in vascular smooth muscle cells, where depolarization with high potassium upregulates the calcium channel a1C subunit (Sonkusare et al., 2006; Pesic et al., 2004), indicating that the long-term regulation of calcium channels may also depend on the cell type. However, in contrast to the abundant knowledge on short-term Ca2+ channel inactivation, long-term regulation of ion channels transcription, translation and expression is not completely understood yet. On the other hand, Ca2+ itself is a main regulator of the expression of many genes and activity-dependent, Ca2+regulated gene expression plays a critical role in diverse neural and neuroendocrine functions, including cell differentiation (Buonanno and Fields, 1999; Gu and Spitzer, 1997), survival (Ghosh et al., 1994), and learning and memory (Svoboda and Mainen, 1999). In chromaffin cells, activitydependent gene regulation has been demonstrated for the catecholamine biosynthetic enzymes phenylethanolamine Nmethyltransferase (Evinger et al., 1994) and tyrosine hydroxylase (Lewis-Tuffin et al., 2004), and for voltage-gated sodium channels (Kobayashi et al., 2002). The work described here was designed to analyze the effects of chronic depolarization on the different calcium channel coding genes expressed in chromaffin cells. With that aim, we have measured the effect of depolarization on Ca2+ channel a1 subunit transcript levels by real time quantitative PCR. We show that chronic depolarization induces a sustained elevation of intracellular Ca2+ which leads to the downregulation of CaV1.1, CaV1.2, CaV1.3, CaV2.2 and CaV2.3 mRNAs, while CaV2.1 transcripts are not affected. The mRNA decrease is followed by a reduction in the whole-cell Ca2+ current amplitude. Additionally, we observed that the depolarizationinduced Ca2+ channel downregulation depends on Ca2+ entry through the L-type VGCC, as it is abrogated by the specific Ltype channel blocker nifedipine. 2. Materials and methods 2.1. Cell isolation and culture Bovine chromaffin cells were isolated as described before (Benavides et al., 2004). Briefly, glands were washed with Ca2+-free Locke’s solution (NaCl
154 mM, KCl 5.6 mM, HNaCO3 3.6 mM, HEPES 10 mM, glucose 5.6 mM, pH 7.4) and incubated in the same solution containing 0.2% collagenase and 0.5% bovine serum albumin for 45 min at 37 8C. Immediately afterwards glands were opened, the medulla separated from the cortex and incubated for an additional 30 min period. The suspension was filtered through a nylon mesh and chromaffin cells were further purified by means of a Percoll gradient. Then cells were washed twice and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and penicillin. Cells were plated at a density of 12 106 cells per T25 culture flask for RNA isolation or 105 cells/ml in glass coverslips for Ca2+ imaging and electrophysiological recordings. Cells were maintained in an incubator at 37 8C in the presence of 5% CO2 until their utilization.
2.2. Treatments One day after plating, the medium was removed, and cells were incubated with fresh medium containing either 5 mM (K5) or 25 mM-KCl (K25) for 24 h. When effects of nifedipine (1 mM), v-conotoxin GVIA (1 mM), v-agatoxin IVA (1 mM) or SNX (400 nM) were examined, blockers were added 1 h before and maintained during the K25 stimulation period. The selection of supramaximal concentration of each blocker, known to fully inhibit its specific channel type, was made on the bases of previous experience from our and other laboratories (Benavides et al., 2005; Aldea et al., 2002; Breustedt et al., 2003; Kohlmeier and Leonard, 2006).
2.3. Reagents Nifedipine and v-agatoxin IVA were provided by Sigma, SNX-482 was from alomone and v-conotoxin GVIA and Fluo-4 acetoxymethyl ester was purchased from Molecular Probes. The other reagents were obtained from Sigma, Panreac or Merck.
2.4. Chromaffin cell viability assessment
2.4.1. LDH assay Chromaffin cell viability was quantified by measuring the LDH released from damaged cells into the bathing medium. CytoTox 96 kit from Promega was used following the manufacturer’s recommendations. Released LDH to the culture medium was measured after 48 h incubation in K25 culture medium. Results are expressed as percentage of the total (released + intracellular) LDH activity. Intracellular LDH was measured after performing two freeze and thaw cycles to disrupt all the cells.
2.5. Hoechst 33342 staining The nuclear morphology of control and 48 h K25-treated chromaffin cells was studied by using the cell-permeable DNA dye Hoechst 33342. Cells with homogeneously stained nuclei were considered to be viable, whereas the presence of chromatin condensation and/or fragmentation was indicative of apoptosis. Hoechst 33342 staining images were obtained by using a fluorescence photomicroscope (Leica DMRXA), with excitation centered at 360 nm, a 400-nm beam splitter, and emission longer than 425 nm. Normal and apoptotic nuclei were counted in three different slides of four independent chromaffin cell cultures.
2.6. RNA isolation and reverse transcription (RT) Total RNA was obtained with the Trizol1 Reagent (Invitrogen) following the manufacturer protocol. The isolated RNA was subsequently treated with DNase (Promega) to remove any contaminating genomic DNA. The integrity of RNA was always checked by running an aliquot in an agarose gel. Reverse transcription was performed using 2 mg of DNase-treated RNA in 20 ml of reaction volume. Previously, 1 ml of 10 mM dNTP mix (Applied Biosystem) and 50 ng of random hexamers (Applied Biosystem) were added to each RNA sample and incubated at 65 8C for 5 min. Then, the samples were incubated with 1 First-Strand Buffer (Invitrogen), 1 ml of 0.1 M dithiothreitol
A. Benavides et al. / Neurochemistry International 50 (2007) 395–403 (InvitrogenTM), 20 units of RNase inhibitor (Applied Biosystem) and 200 units of SuperScriptTM III Reverse Transcriptase (Invitrogen), for 1 h at 50 8C. Samples were stored at 20 8C until their utilization.
2.7. Real-time quantitative PCR using SYBR Green I Changes in the mRNA expression of CaV subunits were examined by real-time quantitative PCR using an ABIPrism 7000 Sequence Detection System (Applied Biosystem). cDNA (1 ml of reverse transcription product) was amplified using SYBR1 Green PCR Master Mix (Applied Biosystems) in the presence of primer oligonucleotides specific for CaV1.1, CaV1.2, CaV1.3, CaV2.1, CaV2.2, CaV2.3 and 18SrRNA. The PCR conditions were as follows: 95 8C for 10 min, followed by 40 cycles consisting of 95 8C for 15 s and 60 8C for 1 min. The quantification was performed by the comparative Ct (cycle threshold) method (Livak and Schmittgen, 2001), using the 18S ribosomal RNA (18SrRNA) expression level as internal control. K25 treatment did not modified the 18SrRNA expression, being the Ct values for control and K25-treated chromaffin cells 9.85 0.64 and 9.79 0.85 (mean S.D., n = 17), respectively. Primers for all target sequences (Table 1) were designed using the computer Primer Express software program specially provided with the 7000 Sequence Detection System (Applied Biosystems). In all the cases only one amplification product was obtained and its identity was confirmed by sequencing.
2.8. Measurement of intracellular Ca2+ levels Changes in free Ca2+ were evaluated using the fluorescent dye Fluo-4 cellpermeant acetoxymethyl (AM) ester essentially as described before (Gee et al., 2000). Chromaffin cells grown on poly-L-lysine-coated glass coverslips were incubated at 37 8C for 1 h in Krebs–HEPES buffer (mM: KCl 5, NaCl 140, MgCl2 1, CaCl2 2.5, HEPES 10 and glucose 11, pH 7.35) containing 5 mM Fluo4-AM. Then, cells were washed in indicator-free medium and incubated for a further 30 min period to allow complete de-esterification of intracellular AM esters. The experiments were performed at room temperature on the stage of a Nikon inverted microscope using oil immersion 40 objective. The fluorescence was measured using a confocal imaging system with an argon ion laser (UltraView LCI system, Perkin-Elmer Life Science) with excitation at 494 nm. Images were acquired with an UltraPix camera and analysed using UltraView LCI software. All measurements were performed after background subtraction (taken from an area in the field of view lacking cells). Little or no run-down of the fluorescent signal was observed during the experiment.
2.9. Electrophysiological recording of Ca2+ currents All voltage clamp data were obtained using the perforated-patch configuration of the whole cell technique as previously described (Calvo et al., 1995) Adrenal chromaffin cells grown on 20-mm diameter coverslips were washed
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twice and maintained in external bathing solution (in mmol/l): NaCl, 140; KCl, 5; CaCl2, 2.5; MgCl2, 1; HEPES, 10; glucose, 10 (pH 7.4). TTX at a final concentration of 1 mM was added to the bathing solution before recording. Recording micropipettes (2–4 MV) were prepared on a P-97 puller (Sutter Instruments, New York, NY, USA). The intracellular pipette solution contained (in mmol/l): 55 CsCl, 50 Cs2SO4, 7 MgSO4, 10 HEPES (pH 7.2). Nystatin at a final concentration of 250 pg/ml was added immediately before the experiment. 24 h K25-treated chromaffin cells were washed and incubated for 1 h in control K5 growth medium for1 h before the electrophysiological recording, in order to remove the voltage inactivation of calcium channels. Channel activity was recorded using an EPC-7 patch-clamp amplifier (HEKA, Lambrecht, Germany) Capacity transients were cancelled, and the pipette potential was set at 70 mV. Ten minutes after gigaseal formation, 10 mV increment depolarizing pulses were applied. Computer analysis of current signals was performed using pClamp 9.0 (Axon Instrument, CA, USA). All experiments were carried out at room temperature.
2.10. Statistics All data were analyzed using the STATGRAPHICS plus 5.1 software package (Rockville, MD, USA). Comparison between two groups (K5 versus K25) was performed using Student’s t-test. One-way ANOVA followed by Fisher’s least significance procedure was applied when multiple comparisons among more than two groups were required; p values less than 0.05 were considered statistically significant.
3. Results 3.1. Chronic depolarization induces sustained Ca2+ increases in chromaffin cells Most studies that have examined mechanisms of activitydependent gene expression have used chronic membrane depolarization induced by elevated K+, to raise intracellular Ca2+ levels. The effects of short KCl treatments on chromaffin cell intracellular Ca2+ have been described previously (Calvo et al., 1995; Neher and Augustine, 1992; Rosario et al., 1989). This type of depolarization causes a rapid increase in intracellular Ca2+ concentration with a steady decline after reaching a peak. In contrast, in the present study we were specifically interested in an experimental model based on prolonged depolarization of chromaffin cells, to induce a maintained raise in intracellular Ca2+ levels. With that aim, chromaffin cells were treated with K25 for 24 h. Intracellular
Table 1 Forward (FWD) and reverse (REV) primer sequences used to amplify the cDNAs of interest and their predicted product length Name
Accession number
Sequence
CaV1.1 (a1S)
AJ621049
CaV1.2 (a1C)
AJ621048
CaV1.3 (a1D)
AJ621050
CaV2.1 (a1A)
AJ621051
CaV2.2 (a1B)
AF173882
CaV2.3 (a1E)
AF244126
18SrRNA
AF176811
FWD: 50 -TCAGCAGTGCCGCCTTG-30 REV: 50 -ACAGAGGTGAACCCGATGTCA-30 FWD: 50 -ACGTGGTCAACTCCACCTACTT-30 REV: 50 -AGCTTCAGGACCATCTCCACTG-30 FWD: 50 -CGTGGTGAACTCCTCGCCT-30 REV: 50 -ACCATGTTCAGAATGTCCATGG-30 FWD: 50 -CGGATGAGTCCAAGGAGTTTG-30 REV: 50 -TCTTCCACTCTCGATCCTTCG-30 FWD: 50 -GACAACGTCGTCCGCAAATAC-30 REV: 50 -ATCTTGATCCCAGCCTCGAA-30 FWD: 50 -CCTTTAAGGCCCTTCCCTATGT-30 REV: 50 -TTGTGCCGGTTGATGTGACT-30 FWD: 50 -CTTTCGAGGCCCTGTAATTGGA-30 REV: 50 -TATTGGAGCTGGAATTACCGCG-30
Product size (bp) 94 169 129 92 206 124 105
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Fig. 1. Chromaffin cells maintained in depolarizing medium exhibit a persistent elevation in the intracellular calcium levels. Chromaffin cells were maintained in either control (K5) or K25 medium for 24 h. Ca2+ levels were measured afterwards using the Ca2+ sensitive fluorescent dye Fluo-4. (A) Representative microphotograph showing the fluorescence emission of control (left) and K25 treated chromaffin cells (right). Images are coloured according to fluorescence intensity, with red representing high Ca2+ concentrations and blue representing low Ca2+ concentrations. (B) Quantitative analysis of six independent experiments performed as described above. Fluorescence intensity, expressed as fluorescence arbitrary units was measured for control (n = 54 cells) and K25 (n = 83 cells). Bars represent the mean S.D. **p < 0.001.
free Ca2+ concentration was measured afterwards, using the fluorescent Ca2+ indicator Fluo-4. Representative images are shown in Fig. 1A. The numerical values obtained for control and K25-treated cells, expressed in relative fluorescence units, were 96 17 and 176 25, respectively (mean standard deviation). These results, presented in Fig. 1B, indicate that cells grown in K25 medium for 24 h retain a significantly elevated intracellular Ca2+ concentration. 3.2. K25 incubation does not affect chromaffin cell viability Although depolarization with KCl 25 mM is very broadly used as a model of chronic depolarization and as survival inducing factor for the culture of some types of neurons, it has been recently reported that similar potassium concentrations can induce calcium dependent chromaffin cell death (CanoAbad et al., 2001). To check if cell death was induced in our experimental conditions we analyzed chromaffin cell viability in 24 h K25-treated cells. Two different methods were used to assess cell viability, the analysis of nuclear morphology by Hoechst staining and the quantification of LDH release. The results, depicted in Fig. 2 shows that no differences were observed between control and K25 treated cells, indicating that under our experimental conditions chromaffin cell viability was completely preserved.
Fig. 2. K25 does not affect chromaffin cell viability. Chromaffin cells were incubated during 24 h with K25 medium and cell viability was evaluated afterwards. (A) Representative microphotography showing the chromaffin cell nuclei stained with Hoechst 33342. Brighter, condensed apoptotic nuclei are labelled with arrows. (B) Graphic representation of apoptotic nuclei present in control (K5) and K25 treated cells. Data are presented as the percentage of total nuclei. (C) Cell damage was also quantified by the LDH release. LDH was measured in the supernatant of 24 h control (K5) or K25 treated cells. Data are presented as the percentage of total (intracellular plus released) LDH. Data in (B) and (C) represent the mean S.D. of at least seven experiments performed in three independent chromaffin cell cultures.
3.3. Effects of chronic depolarization on a1 subunit coding transcripts The effect of depolarization on the calcium channel subunit coding genes was evaluated by real time PCR. We have previously described that chromaffin cells express at least six different a1 subunits: CaV1.1, CaV1.2 and CaV1.3 coding for Ltype, CaV2.1, CaV2.2 and CaV2.3 coding, respectively, for P/Q-, N- and R-types (Benavides et al., 2004). Expression levels of the a1 subunits of those channel types were compared using RNA obtained from control and K25-treated chromaffin cells. Results, depicted in Fig. 3, revealed that most of the subunits analyzed undergo a significant downregulation when exposed to a sustained raise in the intracellular Ca2+ concentration. The most affected subunits were CaV1.1 and CaV2.3, whose expression levels decreased by 70 and 60%, respectively; CaV1.2, CaV1.3 and CaV2.2 were affected in a very similar extent, showing the three of them an approximately 35% decrease. Only the CaV2.1 subunit remained unaffected after 24-h depolarization. Taken together, the PCR results show that chronic depolarization induces a significant downregulation of L-, N- and R-type calcium channel a1 subunits. Consequently, a strong Ca2+ current downregulation should be expected. 3.4. CaV subunits downregulation is followed by the diminution of calcium current amplitude Chromaffin cell Ca2+ inward currents were recorded using the perforated-patch modality of voltage clamp. In order to
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Fig. 3. Effects of depolarization on Ca2+ channels a1 subunit mRNA expression. RNA obtained from control (K5) and K25-treated chromaffin cells was used for quantification of the distinct CaV subunits. CaV1.1, CaV1.2, CaV1.3, CaV2.1, CaV2.2 and CaV2.3 mRNA expression levels were analyzed by reverse transcription followed by quantitative real time PCR. 18SrRNA expression level was used as internal control. Figures represent the percentage of expression relative to the control. Results are expressed as the mean S.D. of at least six experiments performed in different chromaffin cell cultures. **p < 0.001.
remove voltage dependent inactivation, cells were incubated in 5 mM KCl for 1 h before proceeding to path clamp recording. Ca2+ currents were elicited by 200 ms depolarizing pulses from a membrane holding potential of 70 mV. Chromaffin cell depolarization with K25 during 24 h caused a pronounced downregulation of Ca2+ currents (Fig. 4A). The average peak current elicited at 0 mV decreased from 249 46 in control to 107 19 pA in K25-treated cells, which represents a 57% decline in the current amplitude. When the L-type channel blocker nifedipine was added to the cells 1 h before incubation with K25, depolarization-induced Ca2+ current downregulation was partially prevented. The average peak current at 0 mV was, in this case, 190 36 pA, corresponding to a 24% Ca2+ current diminution relative to the control. Current–voltage curves depicting the average peak current values obtained from 17 different recordings each for control, K25- and K25 + nifedipine-treated cells are shown in Fig. 4B.
To determine if the other channel types present in chromaffin cells were also involved in the effect of depolarization on CaV subunit gene expression, cells were treated with the P/Q-type channel blocker v-agatoxin IVA (1 mM); the N-type channel
3.5. Depolarization-induced changes in calcium channel a1 subunit mRNAs are mediated by L-type calcium channels As we described above, the effects of chronic depolarization on Ca2+ currents were partially abrogated by the L-type channel blocker nifedipine. Thus, in the next set of experiments we analyzed the effect of nifedipine on depolarization-induced a1 mRNA changes. Cells were pretreated with nifedipine for 1 h before and during K25 exposure. The results, shown in Fig. 5, indicate that Ca2+ entry through the L-type channels is responsible for the a1 mRNA downregulation in a1 transcripts, as treatment with nifedipine completely avoided K25-evoked changes.
Fig. 4. Depolarization induces calcium current inhibition. Chromaffin cells were incubated in control (K5) or 25 mM KCl containing growth medium for 24 h and Ca2+ currents were recorded afterwards using the perforated patch clamp technique. Holding potential was set at 70 mV and Ca2+ currents were elicited by 200 ms duration/10 mV-increment depolarizing pulses. (A) Representative traces of Ca2+ currents elicited by a depolarizing pulse to 40 and 0 mV from a holding potential of 70 mV in control (K5), 25 mM KCl and 25 mM KCl + 1 mM nifedipine treated cells. (B) I–V relation of peak Ca2+ current obtained from chromaffin cells treated as described above. Results are expressed as the mean S.D. of 17 recordings for each condition performed from three independent cultures.
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Fig. 5. Depolarization-induced calcium channel a1 subunits downregulation depends on calcium influx through L-type calcium channels. Chromaffin cells were exposed to KCl 25 mM for 24 h in the presence or absence of the L-type Ca2+ channel blocker nifedipine. RNA obtained from control (K5), K25-, nifedipine- and K25 + nifedipine-treated cells was used for quantification of the distinct CaV subunits. CaV1.1, CaV1.2 and CaV1.3 (left), CaV2.2 and CaV2.3 (right) were analyzed by quantitative real time PCR using specific oligonucleotides and the 18SrRNA expression level as internal control. Figure represents the percentage of expression relative to the control. Results are expressed as the mean S.D. of at least six experiments performed in different chromaffin cell cultures. **p < 0.001.
blocker v-conotoxin GVIA (1 mM) or the R-type channel blocker SNX (400 nM) for 1 h before and during K25 incubation. As shown in Fig. 6, none of these blockers showed a significant effect on K25-induced changes in a1 subunit transcript levels, suggesting that Ca2+ influx through specifically the L-type channel mediates the a1 mRNA changes induced by chronic depolarisation. 4. Discussion Our results show that chronic depolarization of chromaffin cells causes a severe downregulation of the mRNAs coding for the Ca2+ channels a1 subunit which is followed by a substantial reduction of voltage-sensitive Ca2+ currents. The originality of this work is based in the absence of studies on the longer-term regulation of Ca2+ channels at both the transcriptional and posttranscriptional level, which is surprising considering the important role of Ca2+ itself as a master regulator of transcription. Conversely, the different mechanisms involved in the short-term voltage- and Ca2+-dependent Ca2+ channel inactivation have been extensively described in previous studies (for review, see Budde et al., 2002; Morad and Soldatov, 2005),
In addition, we present here the first evidence that Ca2+ channel mRNA transcripts are downregulated by chronic depolarization. The strategy used was to apply prolonged depolarization to induce persistent intracellular Ca2+ elevation, monitoring the effects of this Ca2+ rise on the genes encoding the Ca2+ channel a1 subunits. Interestingly, this downregulation affects all the a1 subunit types expressed in chromaffin cells, with the exception of the CaV2.1 subunit which remains unmodified. We argue that the observed mRNA downregulation contributes to the Ca2+ current attenuation observed after long-term depolarization, and that this downregulation is triggered as part of a feedback regulatory mechanism which defends cells against excessive, potentially lethal Ca2+ increases. The present study uses adrenal chromaffin cells as an experimental system to study long-term regulation of Ca2+ channels. Electrophysiological and pharmacological studies have demonstrated that these cells express L-, P/Q-, N- and Rtype channels (Albillos et al., 2000), and the fact that all of them contribute to the regulation of catecholamine release (Villarroya et al., 1999) demonstrates that they are physiologically active. In this work we analyze the extent to which Ca2+ channel a1 subunits are affected by sustained intracellular Ca2+
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Fig. 6. Calcium influx through P/Q-, N- and R-type channels is not involved in depolarization-induced calcium channel a1 subunits downregulation. Chromaffin cells were exposed to KCl 5 or 25 mM for 24 h in the presence or absence of the calcium channel blockers v-conotoxin GVIA, v-agatoxin IVA and SNX-482. RNA obtained from those cells was used for quantification of the distinct CaV subunits. CaV1.1, CaV1.2 and CaV1.3 (left), CaV2.2 and CaV2.3 (right) were analyzed by quantitative real time PCR using specific oligonucleotides and the 18SrRNA expression level as internal control. Figure represents the percentage of expression relative to the control values. Results are expressed as the mean S.D. of at least four experiments performed in different chromaffin cell cultures. *p < 0.01; ** p < 0.001.
increase. In preparation to this article, we have recently shown that at least six different genes coding for Ca2+ channel a1 subunits are expressed in chromaffin cells, and are differentially regulated in cultured cells (Benavides et al., 2004). Chronic depolarization has been shown to be a regulatory signal of calcium channel molecules. While it is involved in the hypertension-induced L-type channel a1C subunit upregulation in vascular smooth muscle cells (Sonkusare et al., 2006; Pesic et al., 2004), it has an inhibitory effect in neuronal and neurosecretory cells, downregulating the number of Ca2+ channels (Liu et al., 1995). It reduces dihydropiyridine binding sites and Ca2+ influx in PC-12 (DeLorme et al., 1988; Feron and Godfraind, 1995), GH4C1 cells (Liu et al., 1994) and chick retinal neurons (Ferrante et al., 1991) and diminishes Ca2+ currents in rat myenteric (Fickbohm and Willard, 1999; Franklin et al., 1992) and molluscan neurons (Berdan et al., 1993). In most of those studies the reduction of Ca2+ current was attributed to a diminution in the number of channels, which was caused by either internalization or protein degradation while only one of those reports analyzed the effects of chronic depolarization on the Ca2+ channel subunit transcript levels (Feron and Godfraind, 1995). Therefore, a major interest of our
study was to analyze if coding genes for Ca2+ channel subunit were regulated by chronic depolarization. For this purpose we used real time PCR-based analysis, which allowed us to check if the incubation of chromaffin cells with depolarizing medium alters the transcript levels of the different a1, pore-forming, Ca2+ channel subunits. Among the six different a1 subunit gene products express by chromaffin cells, CaV1.1, CaV1.2 and CaV1.3 form L-type channels, and CaV2.1, CaV2.2 and CaV2.3 correspond to the P/Q-, N- and R-type channels, respectively. Our results revealed that a1 subunit transcripts are strongly downregulated by persistent depolarization. Among the six different subunits analyzed, two of them (CaV1.1 and CaV2.3) were decreased to less that 50% the control values, CaV1.2, CaV1.3 and CaV2.2 were downregulated to around 64% of control values and only one subunit, the CaV2.1, remained unchanged. Therefore, the effect of prolonged depolarization on Ca2+ channel a1 subunit transcripts can be considered very broad and intense, and a likely contributor to the consequences of depolarization on Ca2+ currents. We have used 25 mM KCl to induce a sustained calcium increase. This potassium concentration does not affect the chromaffin cell viability measured by Hoechst staining and
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LDH release. In contrast, similar KCl concentration has been shown to induce chromaffin cell death (Cano-Abad et al., 2001). However, the discrepancies between those results and ours could be explained by the fact that the authors used serumfree Krebs–HEPES solution for their experiments, rendering chromaffin cells more sensitive to damage than in our case, where complete, serum containing, growth medium was used during the 24 h incubation with K25. Finally, we used selective Ca2+ channel blockers to investigate which specific pathways were linked to a1 transcripts regulation. We found that nifedipine, but not vagatoxin IVA, v-conotoxin GVIA or SNX482, avoided the depolarization effects on Ca2+ channel subunit mRNAs, concluding that increased Ca2+ entry through the L-type channel is the mechanism responsible for the CaV1.1, CaV1.2, CaV1.3, CaV2.2 and CaV2.3 mRNA downregulation, whereas Ca2+ influx through the other channel types is not involved in that effect. It is worth noting that, consistently with the previous results, chronic depolarization-induced Ca2+ current downregulation is also overcome by treatment with nifedipine. However, although mRNA downregulation was completely blunted by nifedipine, Ca2+ current was only partially restored, suggesting that depolarization may also regulate calcium channel subunit peptide turnover or targeting to the membrane. Also, interestingly, nifedipine treatment by itself increased the CaV subunits transcript levels, suggesting that the feedback mechanism works in both directions. In agreement with this hypothesis, it has been shown that drugs that decrease excitability of bovine adrenal chromaffin cells by different mechanisms (ethanol, alprazolam, buspirone) produce a marked increase in binding sites for [3H]dihydropyridines on cell membranes (Brennan and Littleton, 1991). There are several potential Ca2+-dependent steps in the process of new gene expression. Ca2+ has been found to regulate the expression of factors related to mRNA transcription, elongation, splicing, stability and translation (West et al., 2001). Therefore, work is underway to determine whether the decrease in mRNA levels that we describe here is due to transcriptional repression of CaV genes or to posttranscriptional mechanisms. In summary, our results indicate a novel mechanism by which depolarization regulates channel function through altering the abundance of CaV a1 transcripts. Since regulation of ionic channels plays a pivotal role in controlling cell signalling and viability, the downregulation of Ca2+ channel genes could be essential for cell physiology, and provide a critical protection to chronically activated cells against Ca2+-mediated damage in neuroendocrine cells. Acknowledgments We want to thank Vanessa Guijarro, Juana Rozalen and Maria Isabel Miquel for their expert technical assistance. This work has been supported, in part, by grants SAF2001-0760 from CICYT and PAI05-017 from JCCM to S.C., D.P. and N.F. are fellows from JCCM.
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A. Benavides et al. / Neurochemistry International 50 (2007) 395–403 Gee, K.R., Brown, K.A., Chen, W.N., Bishop-Stewart, J., Gray, D., Johnson, I., 2000. Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes. Cell Calcium 27, 97–106. Ghosh, A., Carnahan, J., Greenberg, M.E., 1994. Requirement for BDNF in activity-dependent survival of cortical neurons. Science 263, 1618– 1623. Gu, X., Spitzer, N.C., 1997. Breaking the code: regulation of neuronal differentiation by spontaneous calcium transients. Develop. Neurosci. 19, 33–41. Hofmann, F., Lacinova, L., Klugbauer, N., 1999. Voltage-dependent calcium channels: from structure to function. Rev. Physiol. Biochem. Pharm. 139, 33–87. Hong, S.J., Lnenicka, G.A., 1995. Activity-dependent reduction in voltagedependent calcium current in a crayfish motoneuron. J. Neurosci. 15, 3539– 3547. Klugbauer, N., Lacinova, L., Marais, E., Hobom, M., Hofmann, F., 1999. Molecular diversity of the calcium channel alpha2delta subunit. J. Neurosci. 19, 684–691. Kobayashi, H., Shiraishi, S., Yanagita, T., Yokoo, H., Yamamoto, R., Minami, S., Saitoh, T., Wada, A., 2002. Regulation of voltage-dependent sodium channel expression in adrenal chromaffin cells: involvement of multiple calcium signaling pathways. Ann. NY Acad. Sci. 971, 127–134. Kohlmeier, K.A., Leonard, C.S., 2006. Transmitter modulation of spike-evoked calcium transients in arousal related neurons: muscarinic inhibition of SNX482-sensitive calcium influx. Eur. J. Neurosci. 23, 1151–1162. Lewis-Tuffin, L.J., Quinn, P.G., Chikaraishi, D.M., 2004. Tyrosine hydroxylase transcription depends primarily on cAMP response element activity, regardless of the type of inducing stimulus. Mol. Cell. Neurosci. 25, 536–547. Li, M., Jia, M., Fields, R.D., Nelson, P.G., 1996. Modulation of calcium currents by electrical activity. J. Neurophysiol. 76, 2595–2607. Liu, J., Bangalore, R., Rutledge, A., Triggle, D.J., 1994. Modulation of L-type Ca2+ channels in clonal rat pituitary cells by membrane depolarisation. Mol. Pharm. 45, 1198–1206. Liu, J., Rutledge, A., Triggle, D.J., 1995. Short-term regulation of neuronal calcium channels by depolarisation. Ann. NY Acad. Sci. 765, 119–133. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(delta delta C(T)) method. Methods 25, 402–408.
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Lnenicka, G.A., Hong, S.J., 1997. Activity-dependent changes in voltagedependent calcium currents and transmitter release. Mol. Neurobiol. 14, 37–66. Morad, M., Soldatov, N., 2005. Calcium channel inactivation: possible role in signal transduction and Ca2+ signalling. Cell Calcium 38, 223–231. Neher, E., Augustine, G.J., 1992. Calcium gradients and buffers in bovine chromaffin cells. J. Physiol. 450, 273–301. Orrenius, S., Zhivotovsky, B., Nicotera, P., 2003. Regulation of cell death: the calcium-apoptosis link. Nat. Rev. Mol. Cell. Biol. 4, 552–565. Perez-Garcia, M.T., Kamp, T.J., Marban, E., 1995. Functional properties of cardiac L-type calcium channels transiently expressed in HEK293 cells. Roles of alpha 1 and beta subunits. J. Gen. Physiol. 105, 289–305. Peri, R., Triggle, D.J., Singh, S., 2001. Regulation of L-type calcium channels in pituitary GH(4)C(1) cells by depolarisation. J. Biol. Chem. 276, 31667– 31673. Pesic, A., Madden, J.A., Pesic, M., Rusch, N.J., 2004. High blood pressure upregulates arterial L-type Ca2+ channels: is membrane depolarization the signal? Circ. Res. 94, e97–e104. Rosario, L.M., Soria, B., Feuerstein, G., Pollard, H.B., 1989. Voltage-sensitive calcium flux into bovine chromaffin cells occurs through dihydropyridinesensitive and dihydropyridine- and omega-conotoxin-insensitive pathways. Neuroscience 29, 735–747. Sonkusare, S., Palade, P.T., Marsh, J.D., Telemaque, S., Pesic, A., Rusch, N.J., 2006. Vascular calcium channels and high blood pressure: pathophysiology and therapeutic implications. Vascul. Pharm. 44, 131–142. Stotz, S.C., Zamponi, G.W., 2001. Structural determinants of fast inactivation of high voltage-activated Ca(2+) channels. Trends Neurosci. 24, 176–181. Svoboda, K., Mainen, Z.F., 1999. Synaptic [Ca2+]: intracellular stores spill their guts. Neuron 22, 427–430. Villarroya, M., Olivares, R., Ruiz, A., Cano-Abad, M.F., de, P.R., Lomax, R.B., Lopez, M.G., Mayorgas, I., Gandia, L., Garcia, A.G., 1999. Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells. J. Physiol. 516 (Pt 2), 421–432. West, A.E., Chen, W.G., Dalva, M.B., Dolmetsch, R.E., Kornhauser, J.M., Shaywitz, A.J., Takasu, M.A., Tao, X., Greenberg, M.E., 2001. Calcium regulation of neuronal gene expression. Proc. Natl. Acad. Sci. U.S.A. 98, 11024–11031.
Differential regulation of calcium channel coding genes by prolonged depolarization A. Benavides, D. Pastor, N. Fradejas, D. Tornero, S. Calvo * Faculty of Medicine and Regional Center for Biomedical Research, University of Castilla La Mancha, Albacete, Spain Received 18 July 2006; received in revised form 14 September 2006; accepted 18 September 2006 Available online 23 October 2006
Abstract Calcium channels must be subjected to a very precise regulation in order to preserve cell function and viability. Voltage gated calcium channels (VGCC) represent the main pathway for calcium entry in excitable cells. This explains why depolarization induces a rapid-onset and short-term inactivation of calcium currents. Contrarily to this well-documented mechanism to maintain calcium below toxic levels, the regulatory pathways inducing longer-lasting changes and cell surface expression of functional calcium channels are largely unknown. Since calcium is a main player in the activity-dependent regulation of many genes, we hypothesize that calcium channel coding genes could be also subjected to activity-dependent regulation. We have used prolonged depolarization to analyze the effects of sustained intracellular calcium elevation on the mRNAs coding for the different a1 pore-forming subunits of the calcium channels expressed in chromaffin cells. Our findings reveal that persistent depolarization is accompanied by a prolonged intracellular calcium elevation and reduction of calcium current. This calcium current inhibition could be mediated, at least partially, by the downregulation of the mRNAs coding for several a1 subunits. Thus, we show here that depolarization inhibits the expression of CaV1.1, CaV1.2, CaV1.3, CaV2.2 and CaV2.3 mRNAs, while the CaV2.1 mRNA remains unmodified. Moreover, such downregulation of channels depends on calcium entry through the L-type calcium channel, as both mRNA and calcium current changes induced by depolarization are abrogated by L-type channel specific blockers. # 2006 Elsevier Ltd. All rights reserved. Keywords: Calcium channel expression; CaV; Transcription; Activity-dependent gene regulation
1. Introduction Voltage-gated Ca2+ channels (VGCC) are of great importance in coupling excitability to many Ca2+-dependent events within the cells. The Ca2+ channels are complex proteins composed of several (a1, a2d, b and g) subunits. The a1 subunit forms the ionconducting pore and contains the voltage sensor and the interaction sites for channel blockers and activators (Hofmann et al., 1999). The auxiliary subunits a2d, b and g modulate channel properties such as inactivation and channel targeting to the membrane (Klugbauer et al., 1999; Perez-Garcia et al., 1995). Bovine adrenal chromaffin cells express at least four types of voltage-gated Ca2+ channels: L, N, P/Q and R (Aldea et al., 2002) encoded by, at least, six different a1-subunit genes:
* Corresponding author at: Centro Regional de Investigaciones Biome´dicas, Universidad de Castilla La Mancha, Avda. de Almansa 14, 02006 Albacete, Spain. Tel.: +34 967599200x2956; fax: +34 967599324. E-mail address: [email protected] (S. Calvo). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.09.013
CaV1.1, CaV1.2 and CaV1.3 for L-type, CaV2.1, CaV2.2 and CaV2.3 for P/Q-, N- and R-types, respectively (Benavides et al., 2004). These channels coexist in single cells and represent the main physiological trigger for catecholamine release (Aldea et al., 2002). The intracellular Ca2+ concentration must be kept below certain limits to allow cell functionality. It is well known that elevated intracellular Ca2+ concentrations are toxic for the cells and induce cell death (for review, see Orrenius et al., 2003). Moreover, catecholamine secretion by adrenal medulla chromaffin cells must be also strictly regulated to avoid excessive release of catecholamines which could cause irreversible effects such as heart failure, pulmonary edema or hypertension-linked comas (Aunis and Langley, 1999). Ca2+ influx through voltage-activated Ca2+ channels in chromaffin cells must be, therefore, finely tuned. Mammalian cells have developed different mechanisms to avoid maintained or excessive Ca2+ increases. Among them, Ca2+ binding proteins, intracellular reservoirs and extrusion pumps are the most significant. Additionally, VGCC inactivation
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by both, voltage and Ca2+ itself, in a short-term, membranedelimited fashion, also contributes to avoid intracellular Ca2+ overload (reviewed in Budde et al., 2002; Stotz and Zamponi, 2001). There is also compiling evidence pointing at the existence of a longer-lasting Ca2+ channel regulation induced by prolonged depolarization and sustained Ca2+ increases. In this sense, high potassium-induced downregulation of L-type Ca2+ currents has so far been described in myenteric neurons (Franklin et al., 1992); clonal pituitary cells GH4C1 (Liu et al., 1994; Peri et al., 2001); PC12 cells (DeLorme et al., 1988; Feron and Godfraind, 1995); frog skeletal muscle fibers (Carrillo et al., 2004; Escamilla et al., 2001); chick retina and rat cardiac cells (Ferrante et al., 1991). Ca2+ currents have also been reported to decrease in response to chronic electrical stimulation in dorsal root ganglion neurons (Li et al., 1996) and crayfish motoneurons (Hong and Lnenicka, 1995; Lnenicka and Hong, 1997). Opposite effects have been observed in vascular smooth muscle cells, where depolarization with high potassium upregulates the calcium channel a1C subunit (Sonkusare et al., 2006; Pesic et al., 2004), indicating that the long-term regulation of calcium channels may also depend on the cell type. However, in contrast to the abundant knowledge on short-term Ca2+ channel inactivation, long-term regulation of ion channels transcription, translation and expression is not completely understood yet. On the other hand, Ca2+ itself is a main regulator of the expression of many genes and activity-dependent, Ca2+regulated gene expression plays a critical role in diverse neural and neuroendocrine functions, including cell differentiation (Buonanno and Fields, 1999; Gu and Spitzer, 1997), survival (Ghosh et al., 1994), and learning and memory (Svoboda and Mainen, 1999). In chromaffin cells, activitydependent gene regulation has been demonstrated for the catecholamine biosynthetic enzymes phenylethanolamine Nmethyltransferase (Evinger et al., 1994) and tyrosine hydroxylase (Lewis-Tuffin et al., 2004), and for voltage-gated sodium channels (Kobayashi et al., 2002). The work described here was designed to analyze the effects of chronic depolarization on the different calcium channel coding genes expressed in chromaffin cells. With that aim, we have measured the effect of depolarization on Ca2+ channel a1 subunit transcript levels by real time quantitative PCR. We show that chronic depolarization induces a sustained elevation of intracellular Ca2+ which leads to the downregulation of CaV1.1, CaV1.2, CaV1.3, CaV2.2 and CaV2.3 mRNAs, while CaV2.1 transcripts are not affected. The mRNA decrease is followed by a reduction in the whole-cell Ca2+ current amplitude. Additionally, we observed that the depolarizationinduced Ca2+ channel downregulation depends on Ca2+ entry through the L-type VGCC, as it is abrogated by the specific Ltype channel blocker nifedipine. 2. Materials and methods 2.1. Cell isolation and culture Bovine chromaffin cells were isolated as described before (Benavides et al., 2004). Briefly, glands were washed with Ca2+-free Locke’s solution (NaCl
154 mM, KCl 5.6 mM, HNaCO3 3.6 mM, HEPES 10 mM, glucose 5.6 mM, pH 7.4) and incubated in the same solution containing 0.2% collagenase and 0.5% bovine serum albumin for 45 min at 37 8C. Immediately afterwards glands were opened, the medulla separated from the cortex and incubated for an additional 30 min period. The suspension was filtered through a nylon mesh and chromaffin cells were further purified by means of a Percoll gradient. Then cells were washed twice and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and penicillin. Cells were plated at a density of 12 106 cells per T25 culture flask for RNA isolation or 105 cells/ml in glass coverslips for Ca2+ imaging and electrophysiological recordings. Cells were maintained in an incubator at 37 8C in the presence of 5% CO2 until their utilization.
2.2. Treatments One day after plating, the medium was removed, and cells were incubated with fresh medium containing either 5 mM (K5) or 25 mM-KCl (K25) for 24 h. When effects of nifedipine (1 mM), v-conotoxin GVIA (1 mM), v-agatoxin IVA (1 mM) or SNX (400 nM) were examined, blockers were added 1 h before and maintained during the K25 stimulation period. The selection of supramaximal concentration of each blocker, known to fully inhibit its specific channel type, was made on the bases of previous experience from our and other laboratories (Benavides et al., 2005; Aldea et al., 2002; Breustedt et al., 2003; Kohlmeier and Leonard, 2006).
2.3. Reagents Nifedipine and v-agatoxin IVA were provided by Sigma, SNX-482 was from alomone and v-conotoxin GVIA and Fluo-4 acetoxymethyl ester was purchased from Molecular Probes. The other reagents were obtained from Sigma, Panreac or Merck.
2.4. Chromaffin cell viability assessment
2.4.1. LDH assay Chromaffin cell viability was quantified by measuring the LDH released from damaged cells into the bathing medium. CytoTox 96 kit from Promega was used following the manufacturer’s recommendations. Released LDH to the culture medium was measured after 48 h incubation in K25 culture medium. Results are expressed as percentage of the total (released + intracellular) LDH activity. Intracellular LDH was measured after performing two freeze and thaw cycles to disrupt all the cells.
2.5. Hoechst 33342 staining The nuclear morphology of control and 48 h K25-treated chromaffin cells was studied by using the cell-permeable DNA dye Hoechst 33342. Cells with homogeneously stained nuclei were considered to be viable, whereas the presence of chromatin condensation and/or fragmentation was indicative of apoptosis. Hoechst 33342 staining images were obtained by using a fluorescence photomicroscope (Leica DMRXA), with excitation centered at 360 nm, a 400-nm beam splitter, and emission longer than 425 nm. Normal and apoptotic nuclei were counted in three different slides of four independent chromaffin cell cultures.
2.6. RNA isolation and reverse transcription (RT) Total RNA was obtained with the Trizol1 Reagent (Invitrogen) following the manufacturer protocol. The isolated RNA was subsequently treated with DNase (Promega) to remove any contaminating genomic DNA. The integrity of RNA was always checked by running an aliquot in an agarose gel. Reverse transcription was performed using 2 mg of DNase-treated RNA in 20 ml of reaction volume. Previously, 1 ml of 10 mM dNTP mix (Applied Biosystem) and 50 ng of random hexamers (Applied Biosystem) were added to each RNA sample and incubated at 65 8C for 5 min. Then, the samples were incubated with 1 First-Strand Buffer (Invitrogen), 1 ml of 0.1 M dithiothreitol
A. Benavides et al. / Neurochemistry International 50 (2007) 395–403 (InvitrogenTM), 20 units of RNase inhibitor (Applied Biosystem) and 200 units of SuperScriptTM III Reverse Transcriptase (Invitrogen), for 1 h at 50 8C. Samples were stored at 20 8C until their utilization.
2.7. Real-time quantitative PCR using SYBR Green I Changes in the mRNA expression of CaV subunits were examined by real-time quantitative PCR using an ABIPrism 7000 Sequence Detection System (Applied Biosystem). cDNA (1 ml of reverse transcription product) was amplified using SYBR1 Green PCR Master Mix (Applied Biosystems) in the presence of primer oligonucleotides specific for CaV1.1, CaV1.2, CaV1.3, CaV2.1, CaV2.2, CaV2.3 and 18SrRNA. The PCR conditions were as follows: 95 8C for 10 min, followed by 40 cycles consisting of 95 8C for 15 s and 60 8C for 1 min. The quantification was performed by the comparative Ct (cycle threshold) method (Livak and Schmittgen, 2001), using the 18S ribosomal RNA (18SrRNA) expression level as internal control. K25 treatment did not modified the 18SrRNA expression, being the Ct values for control and K25-treated chromaffin cells 9.85 0.64 and 9.79 0.85 (mean S.D., n = 17), respectively. Primers for all target sequences (Table 1) were designed using the computer Primer Express software program specially provided with the 7000 Sequence Detection System (Applied Biosystems). In all the cases only one amplification product was obtained and its identity was confirmed by sequencing.
2.8. Measurement of intracellular Ca2+ levels Changes in free Ca2+ were evaluated using the fluorescent dye Fluo-4 cellpermeant acetoxymethyl (AM) ester essentially as described before (Gee et al., 2000). Chromaffin cells grown on poly-L-lysine-coated glass coverslips were incubated at 37 8C for 1 h in Krebs–HEPES buffer (mM: KCl 5, NaCl 140, MgCl2 1, CaCl2 2.5, HEPES 10 and glucose 11, pH 7.35) containing 5 mM Fluo4-AM. Then, cells were washed in indicator-free medium and incubated for a further 30 min period to allow complete de-esterification of intracellular AM esters. The experiments were performed at room temperature on the stage of a Nikon inverted microscope using oil immersion 40 objective. The fluorescence was measured using a confocal imaging system with an argon ion laser (UltraView LCI system, Perkin-Elmer Life Science) with excitation at 494 nm. Images were acquired with an UltraPix camera and analysed using UltraView LCI software. All measurements were performed after background subtraction (taken from an area in the field of view lacking cells). Little or no run-down of the fluorescent signal was observed during the experiment.
2.9. Electrophysiological recording of Ca2+ currents All voltage clamp data were obtained using the perforated-patch configuration of the whole cell technique as previously described (Calvo et al., 1995) Adrenal chromaffin cells grown on 20-mm diameter coverslips were washed
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twice and maintained in external bathing solution (in mmol/l): NaCl, 140; KCl, 5; CaCl2, 2.5; MgCl2, 1; HEPES, 10; glucose, 10 (pH 7.4). TTX at a final concentration of 1 mM was added to the bathing solution before recording. Recording micropipettes (2–4 MV) were prepared on a P-97 puller (Sutter Instruments, New York, NY, USA). The intracellular pipette solution contained (in mmol/l): 55 CsCl, 50 Cs2SO4, 7 MgSO4, 10 HEPES (pH 7.2). Nystatin at a final concentration of 250 pg/ml was added immediately before the experiment. 24 h K25-treated chromaffin cells were washed and incubated for 1 h in control K5 growth medium for1 h before the electrophysiological recording, in order to remove the voltage inactivation of calcium channels. Channel activity was recorded using an EPC-7 patch-clamp amplifier (HEKA, Lambrecht, Germany) Capacity transients were cancelled, and the pipette potential was set at 70 mV. Ten minutes after gigaseal formation, 10 mV increment depolarizing pulses were applied. Computer analysis of current signals was performed using pClamp 9.0 (Axon Instrument, CA, USA). All experiments were carried out at room temperature.
2.10. Statistics All data were analyzed using the STATGRAPHICS plus 5.1 software package (Rockville, MD, USA). Comparison between two groups (K5 versus K25) was performed using Student’s t-test. One-way ANOVA followed by Fisher’s least significance procedure was applied when multiple comparisons among more than two groups were required; p values less than 0.05 were considered statistically significant.
3. Results 3.1. Chronic depolarization induces sustained Ca2+ increases in chromaffin cells Most studies that have examined mechanisms of activitydependent gene expression have used chronic membrane depolarization induced by elevated K+, to raise intracellular Ca2+ levels. The effects of short KCl treatments on chromaffin cell intracellular Ca2+ have been described previously (Calvo et al., 1995; Neher and Augustine, 1992; Rosario et al., 1989). This type of depolarization causes a rapid increase in intracellular Ca2+ concentration with a steady decline after reaching a peak. In contrast, in the present study we were specifically interested in an experimental model based on prolonged depolarization of chromaffin cells, to induce a maintained raise in intracellular Ca2+ levels. With that aim, chromaffin cells were treated with K25 for 24 h. Intracellular
Table 1 Forward (FWD) and reverse (REV) primer sequences used to amplify the cDNAs of interest and their predicted product length Name
Accession number
Sequence
CaV1.1 (a1S)
AJ621049
CaV1.2 (a1C)
AJ621048
CaV1.3 (a1D)
AJ621050
CaV2.1 (a1A)
AJ621051
CaV2.2 (a1B)
AF173882
CaV2.3 (a1E)
AF244126
18SrRNA
AF176811
FWD: 50 -TCAGCAGTGCCGCCTTG-30 REV: 50 -ACAGAGGTGAACCCGATGTCA-30 FWD: 50 -ACGTGGTCAACTCCACCTACTT-30 REV: 50 -AGCTTCAGGACCATCTCCACTG-30 FWD: 50 -CGTGGTGAACTCCTCGCCT-30 REV: 50 -ACCATGTTCAGAATGTCCATGG-30 FWD: 50 -CGGATGAGTCCAAGGAGTTTG-30 REV: 50 -TCTTCCACTCTCGATCCTTCG-30 FWD: 50 -GACAACGTCGTCCGCAAATAC-30 REV: 50 -ATCTTGATCCCAGCCTCGAA-30 FWD: 50 -CCTTTAAGGCCCTTCCCTATGT-30 REV: 50 -TTGTGCCGGTTGATGTGACT-30 FWD: 50 -CTTTCGAGGCCCTGTAATTGGA-30 REV: 50 -TATTGGAGCTGGAATTACCGCG-30
Product size (bp) 94 169 129 92 206 124 105
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Fig. 1. Chromaffin cells maintained in depolarizing medium exhibit a persistent elevation in the intracellular calcium levels. Chromaffin cells were maintained in either control (K5) or K25 medium for 24 h. Ca2+ levels were measured afterwards using the Ca2+ sensitive fluorescent dye Fluo-4. (A) Representative microphotograph showing the fluorescence emission of control (left) and K25 treated chromaffin cells (right). Images are coloured according to fluorescence intensity, with red representing high Ca2+ concentrations and blue representing low Ca2+ concentrations. (B) Quantitative analysis of six independent experiments performed as described above. Fluorescence intensity, expressed as fluorescence arbitrary units was measured for control (n = 54 cells) and K25 (n = 83 cells). Bars represent the mean S.D. **p < 0.001.
free Ca2+ concentration was measured afterwards, using the fluorescent Ca2+ indicator Fluo-4. Representative images are shown in Fig. 1A. The numerical values obtained for control and K25-treated cells, expressed in relative fluorescence units, were 96 17 and 176 25, respectively (mean standard deviation). These results, presented in Fig. 1B, indicate that cells grown in K25 medium for 24 h retain a significantly elevated intracellular Ca2+ concentration. 3.2. K25 incubation does not affect chromaffin cell viability Although depolarization with KCl 25 mM is very broadly used as a model of chronic depolarization and as survival inducing factor for the culture of some types of neurons, it has been recently reported that similar potassium concentrations can induce calcium dependent chromaffin cell death (CanoAbad et al., 2001). To check if cell death was induced in our experimental conditions we analyzed chromaffin cell viability in 24 h K25-treated cells. Two different methods were used to assess cell viability, the analysis of nuclear morphology by Hoechst staining and the quantification of LDH release. The results, depicted in Fig. 2 shows that no differences were observed between control and K25 treated cells, indicating that under our experimental conditions chromaffin cell viability was completely preserved.
Fig. 2. K25 does not affect chromaffin cell viability. Chromaffin cells were incubated during 24 h with K25 medium and cell viability was evaluated afterwards. (A) Representative microphotography showing the chromaffin cell nuclei stained with Hoechst 33342. Brighter, condensed apoptotic nuclei are labelled with arrows. (B) Graphic representation of apoptotic nuclei present in control (K5) and K25 treated cells. Data are presented as the percentage of total nuclei. (C) Cell damage was also quantified by the LDH release. LDH was measured in the supernatant of 24 h control (K5) or K25 treated cells. Data are presented as the percentage of total (intracellular plus released) LDH. Data in (B) and (C) represent the mean S.D. of at least seven experiments performed in three independent chromaffin cell cultures.
3.3. Effects of chronic depolarization on a1 subunit coding transcripts The effect of depolarization on the calcium channel subunit coding genes was evaluated by real time PCR. We have previously described that chromaffin cells express at least six different a1 subunits: CaV1.1, CaV1.2 and CaV1.3 coding for Ltype, CaV2.1, CaV2.2 and CaV2.3 coding, respectively, for P/Q-, N- and R-types (Benavides et al., 2004). Expression levels of the a1 subunits of those channel types were compared using RNA obtained from control and K25-treated chromaffin cells. Results, depicted in Fig. 3, revealed that most of the subunits analyzed undergo a significant downregulation when exposed to a sustained raise in the intracellular Ca2+ concentration. The most affected subunits were CaV1.1 and CaV2.3, whose expression levels decreased by 70 and 60%, respectively; CaV1.2, CaV1.3 and CaV2.2 were affected in a very similar extent, showing the three of them an approximately 35% decrease. Only the CaV2.1 subunit remained unaffected after 24-h depolarization. Taken together, the PCR results show that chronic depolarization induces a significant downregulation of L-, N- and R-type calcium channel a1 subunits. Consequently, a strong Ca2+ current downregulation should be expected. 3.4. CaV subunits downregulation is followed by the diminution of calcium current amplitude Chromaffin cell Ca2+ inward currents were recorded using the perforated-patch modality of voltage clamp. In order to
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Fig. 3. Effects of depolarization on Ca2+ channels a1 subunit mRNA expression. RNA obtained from control (K5) and K25-treated chromaffin cells was used for quantification of the distinct CaV subunits. CaV1.1, CaV1.2, CaV1.3, CaV2.1, CaV2.2 and CaV2.3 mRNA expression levels were analyzed by reverse transcription followed by quantitative real time PCR. 18SrRNA expression level was used as internal control. Figures represent the percentage of expression relative to the control. Results are expressed as the mean S.D. of at least six experiments performed in different chromaffin cell cultures. **p < 0.001.
remove voltage dependent inactivation, cells were incubated in 5 mM KCl for 1 h before proceeding to path clamp recording. Ca2+ currents were elicited by 200 ms depolarizing pulses from a membrane holding potential of 70 mV. Chromaffin cell depolarization with K25 during 24 h caused a pronounced downregulation of Ca2+ currents (Fig. 4A). The average peak current elicited at 0 mV decreased from 249 46 in control to 107 19 pA in K25-treated cells, which represents a 57% decline in the current amplitude. When the L-type channel blocker nifedipine was added to the cells 1 h before incubation with K25, depolarization-induced Ca2+ current downregulation was partially prevented. The average peak current at 0 mV was, in this case, 190 36 pA, corresponding to a 24% Ca2+ current diminution relative to the control. Current–voltage curves depicting the average peak current values obtained from 17 different recordings each for control, K25- and K25 + nifedipine-treated cells are shown in Fig. 4B.
To determine if the other channel types present in chromaffin cells were also involved in the effect of depolarization on CaV subunit gene expression, cells were treated with the P/Q-type channel blocker v-agatoxin IVA (1 mM); the N-type channel
3.5. Depolarization-induced changes in calcium channel a1 subunit mRNAs are mediated by L-type calcium channels As we described above, the effects of chronic depolarization on Ca2+ currents were partially abrogated by the L-type channel blocker nifedipine. Thus, in the next set of experiments we analyzed the effect of nifedipine on depolarization-induced a1 mRNA changes. Cells were pretreated with nifedipine for 1 h before and during K25 exposure. The results, shown in Fig. 5, indicate that Ca2+ entry through the L-type channels is responsible for the a1 mRNA downregulation in a1 transcripts, as treatment with nifedipine completely avoided K25-evoked changes.
Fig. 4. Depolarization induces calcium current inhibition. Chromaffin cells were incubated in control (K5) or 25 mM KCl containing growth medium for 24 h and Ca2+ currents were recorded afterwards using the perforated patch clamp technique. Holding potential was set at 70 mV and Ca2+ currents were elicited by 200 ms duration/10 mV-increment depolarizing pulses. (A) Representative traces of Ca2+ currents elicited by a depolarizing pulse to 40 and 0 mV from a holding potential of 70 mV in control (K5), 25 mM KCl and 25 mM KCl + 1 mM nifedipine treated cells. (B) I–V relation of peak Ca2+ current obtained from chromaffin cells treated as described above. Results are expressed as the mean S.D. of 17 recordings for each condition performed from three independent cultures.
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Fig. 5. Depolarization-induced calcium channel a1 subunits downregulation depends on calcium influx through L-type calcium channels. Chromaffin cells were exposed to KCl 25 mM for 24 h in the presence or absence of the L-type Ca2+ channel blocker nifedipine. RNA obtained from control (K5), K25-, nifedipine- and K25 + nifedipine-treated cells was used for quantification of the distinct CaV subunits. CaV1.1, CaV1.2 and CaV1.3 (left), CaV2.2 and CaV2.3 (right) were analyzed by quantitative real time PCR using specific oligonucleotides and the 18SrRNA expression level as internal control. Figure represents the percentage of expression relative to the control. Results are expressed as the mean S.D. of at least six experiments performed in different chromaffin cell cultures. **p < 0.001.
blocker v-conotoxin GVIA (1 mM) or the R-type channel blocker SNX (400 nM) for 1 h before and during K25 incubation. As shown in Fig. 6, none of these blockers showed a significant effect on K25-induced changes in a1 subunit transcript levels, suggesting that Ca2+ influx through specifically the L-type channel mediates the a1 mRNA changes induced by chronic depolarisation. 4. Discussion Our results show that chronic depolarization of chromaffin cells causes a severe downregulation of the mRNAs coding for the Ca2+ channels a1 subunit which is followed by a substantial reduction of voltage-sensitive Ca2+ currents. The originality of this work is based in the absence of studies on the longer-term regulation of Ca2+ channels at both the transcriptional and posttranscriptional level, which is surprising considering the important role of Ca2+ itself as a master regulator of transcription. Conversely, the different mechanisms involved in the short-term voltage- and Ca2+-dependent Ca2+ channel inactivation have been extensively described in previous studies (for review, see Budde et al., 2002; Morad and Soldatov, 2005),
In addition, we present here the first evidence that Ca2+ channel mRNA transcripts are downregulated by chronic depolarization. The strategy used was to apply prolonged depolarization to induce persistent intracellular Ca2+ elevation, monitoring the effects of this Ca2+ rise on the genes encoding the Ca2+ channel a1 subunits. Interestingly, this downregulation affects all the a1 subunit types expressed in chromaffin cells, with the exception of the CaV2.1 subunit which remains unmodified. We argue that the observed mRNA downregulation contributes to the Ca2+ current attenuation observed after long-term depolarization, and that this downregulation is triggered as part of a feedback regulatory mechanism which defends cells against excessive, potentially lethal Ca2+ increases. The present study uses adrenal chromaffin cells as an experimental system to study long-term regulation of Ca2+ channels. Electrophysiological and pharmacological studies have demonstrated that these cells express L-, P/Q-, N- and Rtype channels (Albillos et al., 2000), and the fact that all of them contribute to the regulation of catecholamine release (Villarroya et al., 1999) demonstrates that they are physiologically active. In this work we analyze the extent to which Ca2+ channel a1 subunits are affected by sustained intracellular Ca2+
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Fig. 6. Calcium influx through P/Q-, N- and R-type channels is not involved in depolarization-induced calcium channel a1 subunits downregulation. Chromaffin cells were exposed to KCl 5 or 25 mM for 24 h in the presence or absence of the calcium channel blockers v-conotoxin GVIA, v-agatoxin IVA and SNX-482. RNA obtained from those cells was used for quantification of the distinct CaV subunits. CaV1.1, CaV1.2 and CaV1.3 (left), CaV2.2 and CaV2.3 (right) were analyzed by quantitative real time PCR using specific oligonucleotides and the 18SrRNA expression level as internal control. Figure represents the percentage of expression relative to the control values. Results are expressed as the mean S.D. of at least four experiments performed in different chromaffin cell cultures. *p < 0.01; ** p < 0.001.
increase. In preparation to this article, we have recently shown that at least six different genes coding for Ca2+ channel a1 subunits are expressed in chromaffin cells, and are differentially regulated in cultured cells (Benavides et al., 2004). Chronic depolarization has been shown to be a regulatory signal of calcium channel molecules. While it is involved in the hypertension-induced L-type channel a1C subunit upregulation in vascular smooth muscle cells (Sonkusare et al., 2006; Pesic et al., 2004), it has an inhibitory effect in neuronal and neurosecretory cells, downregulating the number of Ca2+ channels (Liu et al., 1995). It reduces dihydropiyridine binding sites and Ca2+ influx in PC-12 (DeLorme et al., 1988; Feron and Godfraind, 1995), GH4C1 cells (Liu et al., 1994) and chick retinal neurons (Ferrante et al., 1991) and diminishes Ca2+ currents in rat myenteric (Fickbohm and Willard, 1999; Franklin et al., 1992) and molluscan neurons (Berdan et al., 1993). In most of those studies the reduction of Ca2+ current was attributed to a diminution in the number of channels, which was caused by either internalization or protein degradation while only one of those reports analyzed the effects of chronic depolarization on the Ca2+ channel subunit transcript levels (Feron and Godfraind, 1995). Therefore, a major interest of our
study was to analyze if coding genes for Ca2+ channel subunit were regulated by chronic depolarization. For this purpose we used real time PCR-based analysis, which allowed us to check if the incubation of chromaffin cells with depolarizing medium alters the transcript levels of the different a1, pore-forming, Ca2+ channel subunits. Among the six different a1 subunit gene products express by chromaffin cells, CaV1.1, CaV1.2 and CaV1.3 form L-type channels, and CaV2.1, CaV2.2 and CaV2.3 correspond to the P/Q-, N- and R-type channels, respectively. Our results revealed that a1 subunit transcripts are strongly downregulated by persistent depolarization. Among the six different subunits analyzed, two of them (CaV1.1 and CaV2.3) were decreased to less that 50% the control values, CaV1.2, CaV1.3 and CaV2.2 were downregulated to around 64% of control values and only one subunit, the CaV2.1, remained unchanged. Therefore, the effect of prolonged depolarization on Ca2+ channel a1 subunit transcripts can be considered very broad and intense, and a likely contributor to the consequences of depolarization on Ca2+ currents. We have used 25 mM KCl to induce a sustained calcium increase. This potassium concentration does not affect the chromaffin cell viability measured by Hoechst staining and
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LDH release. In contrast, similar KCl concentration has been shown to induce chromaffin cell death (Cano-Abad et al., 2001). However, the discrepancies between those results and ours could be explained by the fact that the authors used serumfree Krebs–HEPES solution for their experiments, rendering chromaffin cells more sensitive to damage than in our case, where complete, serum containing, growth medium was used during the 24 h incubation with K25. Finally, we used selective Ca2+ channel blockers to investigate which specific pathways were linked to a1 transcripts regulation. We found that nifedipine, but not vagatoxin IVA, v-conotoxin GVIA or SNX482, avoided the depolarization effects on Ca2+ channel subunit mRNAs, concluding that increased Ca2+ entry through the L-type channel is the mechanism responsible for the CaV1.1, CaV1.2, CaV1.3, CaV2.2 and CaV2.3 mRNA downregulation, whereas Ca2+ influx through the other channel types is not involved in that effect. It is worth noting that, consistently with the previous results, chronic depolarization-induced Ca2+ current downregulation is also overcome by treatment with nifedipine. However, although mRNA downregulation was completely blunted by nifedipine, Ca2+ current was only partially restored, suggesting that depolarization may also regulate calcium channel subunit peptide turnover or targeting to the membrane. Also, interestingly, nifedipine treatment by itself increased the CaV subunits transcript levels, suggesting that the feedback mechanism works in both directions. In agreement with this hypothesis, it has been shown that drugs that decrease excitability of bovine adrenal chromaffin cells by different mechanisms (ethanol, alprazolam, buspirone) produce a marked increase in binding sites for [3H]dihydropyridines on cell membranes (Brennan and Littleton, 1991). There are several potential Ca2+-dependent steps in the process of new gene expression. Ca2+ has been found to regulate the expression of factors related to mRNA transcription, elongation, splicing, stability and translation (West et al., 2001). Therefore, work is underway to determine whether the decrease in mRNA levels that we describe here is due to transcriptional repression of CaV genes or to posttranscriptional mechanisms. In summary, our results indicate a novel mechanism by which depolarization regulates channel function through altering the abundance of CaV a1 transcripts. Since regulation of ionic channels plays a pivotal role in controlling cell signalling and viability, the downregulation of Ca2+ channel genes could be essential for cell physiology, and provide a critical protection to chronically activated cells against Ca2+-mediated damage in neuroendocrine cells. Acknowledgments We want to thank Vanessa Guijarro, Juana Rozalen and Maria Isabel Miquel for their expert technical assistance. This work has been supported, in part, by grants SAF2001-0760 from CICYT and PAI05-017 from JCCM to S.C., D.P. and N.F. are fellows from JCCM.
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