From the Laboratorio de Investigaciones Biomédicas, Departamento de Fisiología and Hospital Universitario Virgen del Rocío, Universidad de Sevilla, E-41013 Seville, Spain
Received for publication, December 10, 2002 , and in revised form, April 4, 2003.
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ABSTRACT |
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INTRODUCTION |
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Despite progress in the understanding of the role of ion channels and gene expression in the cellular responses to hypoxia, long term regulation of ion channel expression by maintained low PO2 is poorly known. There are previous reports showing that prolonged hypoxia down-regulates various voltage-gated K+ (Kv) channel genes in pulmonary artery smooth muscle cells (9), and the opposite has been observed with the Kv1.2 gene in PC12 cells (10). Nevertheless, the effect of chronic hypoxia on voltage-gated ion channel genes has not been systematically addressed, and key questions remain regarding the involvement of the ubiquitous HIF-1-mediated pathway in the modulation by hypoxia of ion channel gene expression.
In this work, we have focused on the regulation by protracted hypoxia of voltage-gated Ca2+ channel genes. These channels are the major pathway for regulated influx of Ca2+ into the cells and play critical roles in diverse cellular processes such as electrical excitability and contraction, hormone secretion, enzyme activity, and gene expression. Importantly, Ca2+ entry through voltage-gated Ca2+ channels is necessary for the acute response to hypoxia of neurosecretory cells (1). There are two major classes of voltage-dependent Ca2+ channels: low voltage-activated or T-type channels and high voltage-activated (HVA) channels, which include the L-, N-, P/Q-, and R-subtypes (11, 12). T-type channels regulate cellular functions susceptible to modulation by low oxygen concentration, such as cellular excitability, differentiation, growth, and proliferation (12). They are predominantly expressed in the G1/S transition stage of the cell cycle (13) as well as in the early stages of differentiation of many embryonic and neonatal tissues (14, 15). In addition, T-type channels are up-regulated by a variety of mitogens and re-expressed in various tissues under proliferative conditions where oxygen supply decreases, such as cardiac cells during heart hypertrophy or cardiomyopathy (16, 17), smooth muscle cells following vascular injury (18), and prostatic tumor cells (19). Based on these precedents, we hypothesized that T-type Ca2+ channels could be a target of the gene expression program developed under hypoxia. We have used for our study the oxygen-sensitive pheochromocytoma-derived PC12 cell line as a model system. These are excitable cells that respond to acute hypoxia with membrane depolarization, increase of extracellular Ca2+ influx, and catecholamine secretion (2022). Chronic hypoxia induces in these cells tyrosine hydroxylase and other genes (23, 24). We show here, both at the molecular and electrophysiological levels, that PC12 cells contain T-type Ca2+ channels and that protracted hypoxia markedly up-regulates their expression. We also present evidence indicating that HIF is involved in this process. Importantly, the low PO2-dependent up-regulation is specific for the T-type Ca2+ channel, since it is barely observed with any of the HVA Ca2+ channels expressed in PC12 cells. These findings define a new role for the T-type Ca2+ channels as regulators of cell excitability and Ca2+ influx during cellular adaptive responses to prolonged hypoxia.
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EXPERIMENTAL PROCEDURES |
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Reverse Transcription and Polymerase Chain ReactionTotal RNA was isolated with Nucleospin RNA II (Macherey-Nagel) following the manufacturer's instructions with the addition of an extra acid phenol/chloroform extraction followed by RNA precipitation. First strand cDNA was synthesized from 24 µg of total RNA using the SuperscriptTM first strand synthesis system for reverse transcriptase-PCR (Invitrogen) with random primers according to the manufacturer's directions. To analyze the expression of T-type channels in PC12 cells, the PCRs were usually carried out for 30 cycles of 96 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min with 2 µl of cDNA. The primers used were as follows: rat 1H, forward (5'-GGCGTGGTGGTGGAGAACTT-3') and reverse (5'-GATGATGGTGGGATTGAT-3') (GenBankTM accession number AF290213
[GenBank]
); rat
1G, forward (5'-ACCTGCCTGACACTCTGCAG-3') and reverse (5'-GCTGGCCTCAGCGCAGTCGG-3') (GenBankTM accession number AF290212
[GenBank]
); and rat
1I, forward (5'-GAGTTAGACAAGCTCCCAGA-3') and reverse (5'-CAGTTGAGGAAGATAAAGGC-3') (GenBankTM accession number AF290214
[GenBank]
). The PCR products were subcloned in pGemT or pBluescript vectors, and their identities were confirmed by complete DNA sequencing.
Northern Blot AnalysismRNA was isolated from total RNA with NucleoTrap mRNA minikit (Macherey-Nagel) following manufacturer's instructions. Equal amounts (5 µg) of mRNA were analyzed by Northern blots according to standard procedures for separation using 1% agarose gel containing formaldehyde (25). RNA from gels was transferred to BrightStar-Plus positively charged nylon membranes (Ambion) and UV-cross-linked prior to hybridization. Labeling of radioactive DNA probes was performed using [32P]dCTP and RediprimeTM II random prime labeling system (Amersham Biosciences). The probes were subsequently purified with MicrospinTM S-400 HR columns (Amersham Biosciences). Hybridization was carried out overnight at 42 °C with Ultrahyb hybridization buffer (Ambion), after which the membranes were washed twice to a stringency of 2x SSC, 0.1% SDS at 55 °C for 15 min followed by three 5-min washes with 0.5x SSC, 0.1% SDS at 55 °C. RNA loading on gels was monitored by ethidium bromide staining and by probing with cyclophilin as a control. Autoradiography was carried out at 80 °C with intensifying screens. Rat cDNA fragments corresponding to nucleotides 47195189 of the 1H subunit and nucleotides 35684426 of the
1G subunit were used to make the radioactive probes. RNA bands on the autoradiographs were quantified using a CanoScan N650U scanner and National Institutes of Health software.
Real Time PCRTotal RNA was isolated from cells after the different treatments, and 4 µg were subsequently used for cDNA synthesis as described above. Real time PCR was performed in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) using SYBR Green PCR Master mix (Applied Biosystems) and the thermocycler conditions recommended by the manufacturer. PCRs were performed in duplicates in a total volume of 30 µl containing 1 or 2 µl of the reverse transcriptase reaction. Each sample was analyzed for -actin to normalize for RNA input amounts and to perform relative quantifications. Primers were designed using the computer program Primer Express (Applied Biosystems). Primers (forward 5'-ACTTGGCCATCGTCCTCCTA-3' and reverse 5'-GCGGCGTTCATCTCAATCTC-3') were generated to the rat
1H subunit of T-type Ca2+ channels and used to amplify a 64-base pair fragment. For the
-subunits of the HVA Ca2+ channels, primers were as follows: forward (5'-TCATCTTCAGCCCAAACAACAG-3') and reverse (5'-TTGGTGAAGATCGTGTCATTGAC-3') for the
1C subunit (GenBankTM accession number M67516
[GenBank]
); forward 5'-AATGCCCTGCTCCAGAAAGA-3' and reverse 5'-CAGACGCTGCCCTAGGTAAGG-3' for the
1B subunit (GenBankTM accession number M92905
[GenBank]
); and forward 5'-GATGGCTCAAGAAAGCAGCAT-3' and reverse 5'-GGCTCCAGGTACCAGTCTTCTG-3' for the
1A subunit (GenBankTM accession number M64373
[GenBank]
). Melting curve analysis showed a single sharp peak with the expected Tm for all samples.
Western BlottingCells were homogenized in lysis buffer containing 50 mM Hepes-KOH, pH 7.3, 250 mM NaCl, 5 mM EDTA, 0.2% Nonidet P-40, 5 mM dithiothreitol, and protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 µg/ml aprotinin, 10 µg/ml pepstatin, 10 µg/ml leupeptin) for 10 min at 4 °C. After centrifugation at 8,000 x g for 5 min, the supernatants were recovered, and protein concentrations were determined with Bradford protein assay reagent. Lysates (20 µg) were loaded and resolved on 6% SDS-polyacrylamide gel electrophoresis followed by transfer to Immun-Blot polyvinylidene difluoride membrane (Bio-Rad). Membranes were probed with 1:500 anti-HIF 2 (Novus-Biologicals) or 1:1000 anti-
-tubulin antibodies (Sigma) and developed with the enhanced chemiluminescence plus Western blotting detection system (Amersham Biosciences).
Antisense Oligonucleotide TreatmentPhosphorothioate oligonucleotides for HIF-2 depletion experiments were as follows: antisense, 5'-CCTCAGCTCCGAGCTGCTC-3'; sense, 5'-GAGCAGCTCGGAGCTGAGG-3'. PC12 cells (
1.5 x 106) were transfected with 200 nM either sense or antisense oligonucleotide with oligofectamine transfection reagent according to the manufacturer's protocol (Invitrogen). 72 h after transfection, treated cells were exposed to hypoxia or normoxia for 12 h, and afterward, cells were lysed, and total RNA and protein were extracted.
1H and HIF 2
mRNA levels were quantified by real time PCR as described above. The primers used for HIF-2
mRNA quantification were as follows: 5'-GCAGATGGATAACTTGTACCTGAAAG-3' and 5'-CTGACAGAAAGATCATATCACCGTCTT-3'.
5'-Flanking Sequences AnalysisThe 5' upstream sequences of the rat, mouse, and human 1H genes were obtained from the Ensembl software system (available on the World Wide Web at www.ensembl.org). The rat
1H gene (GenBankTM accession number AF290213
[GenBank]
) is located in sequence RNOR01500654 on chromosome 10. The mouse
1H gene (GenBankTM accession number NM_021415
[GenBank]
) is located in sequence 17.2400000125000000 on chromosome 17. The human
1H gene (GenBankTM accession number NM_021098
[GenBank]
) is located in sequence AC120498
[GenBank]
.2.1.195680 on chromosome 16.
Electrophysiological RecordingsMacroscopic Ca2+ currents were recorded using the whole cell configuration of the patch clamp technique as adapted to our laboratory (26, 27). Patch electrodes (23 megaohms) were pulled from hematocrit capillary glass (Hirschmann Laborgerate; 1.51.6-mm OD), fire-polished on an MF-830 microforge (Narishige), and coated with silicone elastomer (Sylgard 184; Dow Corning) to decrease capacitance. Voltage clamp recordings were obtained with an EPC-8 patch clamp amplifier (Heka Elektronik) using standard voltage clamp protocols designed with Pulse software (Heka Elektronik). Unless otherwise noted, holding potential was 80 mV. Data were filtered at 10 kHz, digitized at a sampling interval of 20 µs with an ITC-16 A/D converter (Instrutech), and stored on a Macintosh computer. Off-line analysis of data was performed using custom software and Pulse Fit (Heka Elektronik). All experiments were conducted at room temperature, 2224 °C. For whole cell patch recordings, the internal solution contained 110 mM CsCl, 30 mM CsF, 10 mM EGTA, 10 mM HEPES, and 4 mM Mg-ATP; pH was adjusted with CsOH to 7.2, and osmolality was 285 mOsM/kg. The standard bath solution contained 140 mM N-methyl-D-glucamine, 9 mM BaCl2, 1 mM CaCl2, 10 mM HEPES, and 10 mM glucose. pH was adjusted with HCl to 7.4, and osmolality was 300 mOsM/kg.
Statistical AnalysisData were analyzed using Student's t test for unpaired observations with the SigmaPlot program (Jandel). Values are given as mean ± S.E. p values less than 0.05 were considered as statistically significant.
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RESULTS |
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The presence of functional T-type Ca2+ channels in PC12 cells was confirmed by analysis of the total Ca2+ currents recorded with the whole cell configuration of the patch clamp technique. Fig. 1C shows a representative example of Ca2+ current recorded in a PC12 cell during a depolarizing pulse to +20 mV followed by repolarization to 70 mV. Consistent with other analyses on Ca2+ currents in PC12 cells (28, 29), the recorded current appeared to result predominantly from the expression of HVA and fast deactivating channels, as evidenced by the fast tail current generated when the membrane was repolarized to 70 mV. The apparent activation threshold of the whole cell Ca2+ current in PC12 cells was observed at about 50 mV, and maximal current amplitude was observed at +20 mV (see below). However, a slow component in the deactivation, or closing, tail current (indicated by the small arrowheads in Fig. 1, C and D) was consistently observed, suggesting the presence of a population of T-type Ca2+ channels, which are known to close about 1020 times more slowly than HVA channels (12, 27, 3234). The calcium tail currents evoked upon repolarization were analyzed in more detail (Fig. 1D). To quantify the closing time constant (
), we fitted single and double exponential functions to the deactivating segment of the current (top and bottom panels in Fig. 1D). Analysis of the data revealed that the deactivating current is well fitted by a double but not by a single exponential function; therefore, two time constants (
fast and
slow) were estimated (27, 32, 33). At 70 mV, average
fast and
slow values were 0.11 ± 0.005 and 1.34 ± 0.09 ms, respectively (n = 45 cells). These data are compatible with the existence of two populations of Ca2+ channels in PC12 cells, HVA channels that deactivate rapidly, and T-type channels that deactivate slowly. Significantly, our data are in close agreement with the reported electrophysiological parameters for the recombinant rat
1H gene (30). The presence of T-type Ca2+ currents in PC12 cells was further confirmed by their rapid inactivation, another distinct feature of T-type Ca2+ channels (27, 32, 35, 36). Fig. 1E shows representative examples of the deactivating currents recorded in PC12 cells upon repolarization of the cell to 70 mV after a depolarizing pulse to +20 mV lasting either 10 ms (short pulse) or 50 ms (long pulse). The experiments clearly indicated that the slow component of the tail current present at the end of 10-ms pulses disappeared almost completely at the end of the 50-ms pulses, suggesting inactivation of the T-type channels during the maintained depolarization. Therefore, Ca2+ currents in PC12 cells possess a slowly deactivating and fast inactivating component typical of T-type Ca2+ channels. The molecular and the electrophysiological data indicate that
1H is the major T-type Ca2+ channel subunit functionally expressed in PC12 cells.
Selective Induction of T-type Ca2+ Channel Gene Expression by HypoxiaNorthern blot and quantitative real time PCR analyses were performed to determine whether the 1H gene expression is regulated by hypoxia. Fig. 2A shows Northern blot data obtained on PC12 cells after exposure to reduced PO2 (3% oxygen) for 1, 6, 12, and 24 h. In all our experiments, sensitivity of PC12 cells to hypoxia was confirmed by the induction of tyrosine hydroxylase, a well characterized hypoxia-responsive gene (23, 24). The data revealed that hypoxia greatly stimulates
1H mRNA expression in PC12 cells in a time-dependent manner. Six hours of exposure to hypoxia induced a clear increase of the
1H mRNA level, which was potentiated after 12 and 24 h of treatment. Remarkably, the induction of the
1H subunit by hypoxia appeared to be much higher than that observed for the tyrosine hydroxylase gene. To further confirm the results, real time PCR experiments were performed, and the data were compared with those obtained in the Northern blot analysis. The results from at least five independent experiments for each duration of hypoxic treatment are shown on Fig. 2B. Both Northern blot and real time PCR analyses yielded similar results:
5-fold accumulation of the
1H mRNA after 6 h of exposure to low PO2 and a maximal effect at 1224 h (
8-fold induction). We next exposed PC12 cells to a range of oxygen levels from 21% (normoxia) to 3% for 12 h and quantified the amount of
1H mRNA by both Northern blot analysis and real-time PCR. As shown in Fig. 2C, the effect of hypoxia was dose-dependent, being weakly detectable at 12 and 6% oxygen (
1.5- and
2.2-fold induction, respectively) and reaching a maximal induction (
58-fold) at 3%. Oxygen concentrations below 3% were not tested due to the high level of cellular death observed. These results indicate that the
1H T-type Ca2+ channel gene is up-regulated by hypoxia in a time- and dose-dependent manner.
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The data described above prompted us to determine whether up-regulation of gene expression by low PO2 was selective for the T-type Ca2+ channel or whether hypoxia also altered the expression of other Ca2+ channel classes. Fig. 3 shows real time PCR quantification after exposure to 3% oxygen for 12 or 24 h of the mRNA levels of the 1C,
1B, and
1A channel subunits that form, respectively, the L-, N-, and P/Q-types of HVA channels previously described in PC12 cells (28, 29). Comparison of the threshold cycles of the PCR reveals that the amounts of the three mRNAs are slightly but not significantly increased after 12 or 24 h of hypoxia. Thus, in our experimental conditions, chronic hypoxia has little effect on the expression of HVA Ca2+ channels, whereas it is a powerful and selective stimulus for T-type Ca2+ channel expression.
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Hypoxia Increases the Density of Functional T-type Ca2+ ChannelsElectrophysiological experiments were performed to investigate the effect of hypoxia on the level of functional T-type Ca2+ channels in PC12 cells. For these experiments, PC12 cells were either exposed to 3% oxygen for 1820 h or incubated under normoxic conditions. Peak inward current density measured at the end of 10-ms pulses to +20 mV showed no differences in the amount of Ca2+ current generated in normoxia (8.6 ± 0.6 pA/pF, n = 45) and hypoxia (8.0 ± 0.6 pA/pF, n = 42). The average I/V relationship was also slightly altered by low PO2 with only an increase in the density of inward currents in the range between 50 and 10 mV (Fig. 4A). Despite the smallness of Ca2+ currents at these voltages, this last observation suggested that the density of functional T-type Ca2+ channels had increased by hypoxia. The effect of maintained hypoxia was better appreciated by the analysis of the inward tail currents. Fig. 4, B and C, shows the Ca2+ currents generated by step depolarization in a representative PC12 cell exposed to hypoxia for 20 h. After this treatment, there was a characteristic appearance of a large, slowly deactivating component in the tail current (Fig. 4B) and the partial inactivation of the current during long lasting pulses (Fig. 4C), suggesting a high presence of T-type Ca2+ channels in the cells. As shown above for control cells (see Fig. 1E), the slow component of the tail currents, reflecting the closure of the T-type channel population, was also markedly reduced after long lasting pulses in hypoxia-treated cells (Fig. 5, A and B). Shown in Fig. 5, C and D, are frequency histograms of the amplitude of the slowly deactivating current component expressed as percentage of the total tail current. These data show that exposure to hypoxia increased the population of cells with a large component of slowly deactivating tail current; in some cells, the amplitude of the slowly deactivating current was more than half of the total tail current. Although chronic hypoxia increased the expression of T-type channels, the values of the fast and slow closing time constants of the current measured at 70 mV remained unchanged (fast = 0.12 ± 0.01 and
slow = 1.37 ± 0.06 ms, n = 42 cells; p > 0.05 in the two cases when compared with the respective values in control cells). The slowly deactivating component of the tail currents induced by hypoxia was selectively reduced in amplitude by application of 50 µM nickel to the external solution (Fig. 5, E and F), thus further indicating that this component of the Ca2+ current represented the activity of T-type Ca2+ channels (37).
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Induction of T-type Ca2+ channel expression by chronic hypoxia is further illustrated in Fig. 6. The average current density due to T-type Ca2+ channels measured during repolarizations from +20 to 70 mV (3.2 ± 0.47 pA/pF, n = 45 cells) increased almost 2.5 times upon exposure of the cells to hypoxia (7.4 ± 1.4 pA/pF, n = 42 cells) (Fig. 6A). In contrast, the same treatment produced no effect on, or even a slight reduction in, the current density mediated by the fast deactivating Ca2+ channels (Fig. 6B). Accordingly, the ratio of slow/fast deactivating current densities measured in each of the cells was markedly increased in hypoxia (Fig. 6C). These results indicate that, in accord with the up-regulation of 1H mRNA expression detected by molecular biology, protracted hypoxia induces a significant increase in the number of functional T-type Ca2+ channels in PC12 cells.
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T-type Ca2+ Channel Induction Depends on HIF ActivationIt is well known that the effects of hypoxia on gene expression are mimicked by agents such as cobalt chloride and the iron chelator desferrioxamine, which act as nonspecific inhibitors of the prolyl hydroxylases that regulate the protea-somal degradation of HIF isoforms (HIF-1 and HIF-2
) (47, 38, 39). As shown in Fig. 7, A and B, Northern blot and real time PCR experiments revealed that treatment of PC12 cells with CoCl2 or desferrioxamine for 12 h resulted in the accumulation of the
1H mRNA to a degree almost similar to that produced by the hypoxic stimulation. We also tested the effects of dimethyloxalylglycine on
1H mRNA expression. This is a cell-permeant analog of oxoglutarate that competitively inhibits prolyl hydroxylases and consequently stabilizes HIF and up-regulates under normoxic conditions the expression of hypoxia-inducible genes (6, 40). Fig. 7 (A and B) also shows that the amount of
1H mRNA increased after dimethyloxalylglycine treatment. These results strongly suggest that, as it occurs for other genes (15), HIF is involved in the hypoxic up-regulation of T-type Ca2+ channel expression.
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To determine whether HIF is indeed functionally involved in the hypoxic up-regulation of T-type Ca2+ channel expression, we exposed cells to hypoxia once they had been incubated with antisense HIF oligonucleotides (41, 42). In these experiments, we used antisense HIF-2 oligonucleotides, since this appears to be the HIF isoform most actively induced by hypoxia in PC-12 cells (43, 44). As described before (45), HIF-2
is strongly induced by hypoxia at the level of protein but only slightly at the level of mRNA (Fig. 8A). However, incubation with anti-sense HIF-2
oligonucleotides resulted in a marked decrease in the HIF-2
mRNA and protein induced by hypoxia in these cells (Fig. 8A). In fair agreement with these data, antisense inhibition of HIF-2
strongly decreased the hypoxic induction of the
1H subunit, reducing the mRNA levels to values close to those seen in normoxia. Antisense HIF-2
oligonucleotides had no effect on the levels of the T-type channel mRNA in basal conditions (Fig. 8B). In control experiments, sense HIF-2
oligonucleotides showed no effect on the hypoxic induction of
1H mRNA. The involvement of HIF in the hypoxic up-regulation of the
1H Ca2+ channel suggested by these experiments is further supported by the presence of hypoxia-responsive elements in the 5'-flanking region of the
1H gene. We identified numerous sequences compatible with hypoxia-responsive elements (44, 4649) in a region of
1300 bp upstream of the coding sequence of the rat
1H gene. This region is highly conserved among mammals, with more than 71% similarity between rodents and humans and 93% similarity between rats and mice. In Fig. 8C, we represent selected fragments of the aligned rat, mouse, and human sequences, showing six putative HIF consensus DNA binding sites. The core motifs are in boldface type and overlined by arrows to indicate the plus (right arrow) or minus (left arrow) DNA strand location.
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Dependence on Ca2+ of the Regulation of T-type Ca2+ Channel Gene Expression by HypoxiaOne of the earliest known responses to hypoxia in PC12 cells is membrane depolarization and increase of intracellular Ca2+ levels (20). Previous reports have shown that increase of intracellular free Ca2+ is required for the expression of hypoxia-inducible genes, including tyrosine hydroxylase, in PC12 cells (24, 50). To test whether the induction of 1H expression by hypoxia is Ca2+-dependent, PC12 cells were incubated in Ca2+-free medium supplemented with 1 mM EGTA and exposed to normoxia or hypoxia (3% oxygen) for 12 h. Fig. 9 (A and B) shows that the accumulation of
1H mRNA induced by hypoxia is maintained in the absence of extracellular Ca2+. Thus, an increase in intracellular free Ca2+ does not seem to be required for induction of
1H gene expression by hypoxia in PC12 cells. In contrast, it seems that maintained Ca2+ influx might even down-regulate
1H expression.2
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DISCUSSION |
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Among the three -subunits (
1H,
1G, and
1I) of T-type channels already cloned (30, 31, 52, 53),
1H (Cav3.2 gene) is the most abundantly expressed in PC12 cells. The
1G mRNA is detectable only by PCR, and the
1I is undetectable. Accordingly, the electrophysiological data reveal that besides a large population of fast deactivating, or closing, HVA Ca2+ channels, PC12 cells also express a smaller population of slowly deactivating and fast inactivating T-type Ca2+ channels. Significantly, the estimated deactivation time constant of the native T-type Ca2+ channels in PC12 cells is similar to the reported value for the recombinant rat
1H subunit, which has been shown to display faster closing kinetics than the
1G and
1I subunits (30). To our knowledge, T-type Ca2+ channels had been documented in PC12 cells that have undergone neuroendocrine differentiation (54) but not in undifferentiated PC12 cells. The very scant level of expression and the relatively fast deactivation kinetics of the
1H subunit have probably hampered the separation of the T-type Ca2+ channels from the fast deactivating HVA channels in previous electrophysiological studies on PC12 cells. Likewise, T-type Ca2+ channels have not been detected in chromaffin cells until recently by using molecular approaches and analysis on different stages of cellular development. Remarkably, the T-type channels expressed in rat chromaffin cells share properties with the
1H subunit (55). These data are in good agreement with studies showing that the
1H subunit is mostly expressed in peripheral tissues (53), in contrast with
1G and
1I subunits that are more abundant in the brain (52, 56).
Our results clearly show that the 1H T-type Ca2+ channel is overexpressed in chronic hypoxia. This was demonstrated by accumulation of the specific mRNA and by an increase in the membrane current density with kinetic and pharmacological (nickel inhibition) features characteristic of this class of T-type channels (12, 27, 3234, 37). Induction of the
1H subunit by hypoxia was time- and dose-dependent, reaching maximal levels (about 58-fold) after 1224 h of exposure to 3% oxygen. The same hypoxic treatment produced only a 2.5-fold increase in the amplitude of T-type channel current, possibly because these channels are also subjected to postranscriptional regulation by hypoxia and other signaling variables (57). Interestingly, hypoxia has not a marked effect on any of the HVA Ca2+ channel genes expressed in PC12 cells. Neither significant accumulation of the
1C (L-type),
1B (N-type), and
1A (P/Q-type) mRNAs nor significant increase of the fast deactivating Ca2+ mean current density is observed in PC12 cells exposed to hypoxia. On the contrary, a small but significant decrease in the HVA channel mean current density was observed upon hypoxia exposure of the PC12 cells. Although our molecular, electrophysiological, and pharmacological data suggest that the effect of hypoxia is rather selective for a subtype of T-type Ca2+ channels, there is a previous report showing that chronic hypoxia increases the amplitude of Ca2+ currents in PC12 cells (58). In these last experiments, however, the hypoxia challenge was very mild (10% oxygen), and T-type channels were not studied with electrophysiological or molecular techniques. Induction of T-type Ca2+ channels genes by hypoxia is not a singular property of our PC12 cells but a rather general phenomenon, since it has also been observed in adrenal chromaffin cells as well as in primary cultures and clonal cell lines of aortic smooth muscle.3
Induction of T-type Ca2+ channel expression by low PO2 has the same general features (O2 levels, time course, etc.) of the classical hypoxia-inducible genes such as erythropoietin or vascular endothelial growth factor (15). Although there exists the possibility that hypoxia-induced up-regulation of the 1H Ca2+ channel subunit depends on general changes in the cells (i.e. cytoplasmic acidification and subsequent activation of immediate early response genes), our experiments strongly suggest that specific activation of the HIF pathway mediates this effect. Induction of T-type Ca2+ channel gene expression by low PO2 was replicated by cobalt chloride, the iron quelant desferrioxamine, and dimethyloxalylglycine (4, 5, 39, 40). These compounds have been shown to mimic hypoxia by inhibiting an oxygen-, Fe2+-, and oxoglutarate-dependent dioxygenase that under normoxic conditions hydroxylates specific proline and asparagine residues in HIF prior its degradation (68). Moreover, we have shown using antisense HIF-2
oligonucleotides that functional inhibition of HIF-2
protein accumulation reverses the effect of hypoxia on T-type Ca2+ channel expression. This experimental observation is in fair agreement with the existence in the 5'-flanking region of the
1H gene of several putative HIF consensus DNA binding sites (4649). We have also investigated whether Ca2+ is critical for the hypoxiainduced regulation of
1H Ca2+ channel expression. Interestingly, the increase in
1H mRNA induced by hypoxia was completely unaffected by the removal of extracellular Ca2+. This finding contrasts with the observed Ca2+ dependence of the hypoxic induction of some genes in PC12 cells (24, 50). However, recent data demonstrate that intracellular Ca2+ operates through a HIF-1
-independent signaling pathway to activate transcription of hypoxia-inducible genes (59).
The information available on the induction of ion channel coding genes by hypoxia is very scant, and to our knowledge the participation of HIF-dependent mechanisms in the regulation of these genes has not been previously studied. Chronic hypoxia has been reported to decrease the mRNA and protein levels of the K+ channels Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3 in pulmonary arterial myocytes but not in the mesenteric artery (9, 60). Although the role of HIF in the regulation of these Kv genes was not documented, the facts that the down-regulation is constrained to specific arteries and occurs after very prolonged periods of hypoxia (6072 h) suggest a HIF-independent mechanism. In addition, an opposite effect of hypoxia, selective increase of the Kv1.2 gene expression, has been described in PC12 cells (10). Again, the possible participation of HIF in the hypoxic regulation was not addressed in the latter report.
We focused on voltage-dependent T-type Ca2+ channels, because Ca2+ influx through these channels might have a major role in the adaptive cellular responses to prolonged hypoxia. The increase of cell excitability resulting from T-type Ca2+ channel overexpression (30) may be of importance in the control of secretion in chronically hypoxic cells. Accordingly, it has been reported that chronic hypoxia increases free intracellular Ca2+ and enhances the secretory response of PC12 cells to acute hypoxia (21). Chronic intermittent exposures to low PO2 have been shown to increase the excitability of carotid body and sympathoadrenal tissues (61). Besides the possible role of T-type channels in the modulation of cell excitability, they have been suspected of participating in cell cycle progression and proliferation, although the evidence is still being debated (62). It is therefore possible that Ca2+ entry through T-type Ca2+ channels is implicated in the cellular proliferation that usually occurs after acute or chronic hypoxia damage and in the hypoxic environment of proliferating tumor cells. In fact, a recent report has revealed that the 1H T-type Ca2+ channels are overexpressed in human prostate cancer cells in their more aggressive and invasive stages (19). Extreme hypoxia is a hallmark of solid tumors that leads to phenotypic alterations promoting tumor growth and progression (63). A potential role of T-type Ca2+ channels in malignant cellular proliferation would offer a new molecular target that could be exploited therapeutically. Therefore, it will be of particular interest to assess the role of T-type Ca2+ channels in cell growth and tumor biology.
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FOOTNOTES |
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These authors contributed equally to this work.
¶ Present address: Servicio de Otorrinolaringología, Hospital Universitario Central de Asturias, C/Celestino Villamil s/n E-33006, Oviedo, Spain.
Recipient of the "Ayuda a la investigación 2000" of the Juan March Foundation. To whom correspondence should be addressed: Laboratorio de Investigaciones Biomédicas, Edificio de Laboratorios, 2a Planta, Hospital Universitario Virgen del Rocío, Avenida Manuel Siurot s/n, E-41013, Seville, Spain. Tel.: 34-955-012648 or 34-955-013157; Fax: 34-954-617301; E-mail: lbarneo{at}us.es.
1 The abbreviations used are: HIF, hypoxia-inducible transcription factor; Kv, voltage-gated K+; HVA, high voltage-activated; pF, picofarads.
2 R. Del Toro, K. L. Levitsky, J. López-Barneo, and M. D. Chiara, unpublished results.
3 J. Navarro-Antolín, K. L. Levitsky, and J. López-Barneo, unpublished results.
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ACKNOWLEDGMENTS |
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