Induction of T-type Calcium Channel Gene Expression by Chronic Hypoxia*

Cellular responses to hypoxia can be acute or chronic. Acute responses mainly depend on oxygen-sensitive ion channels, whereas chronic responses rely on the hy-poxia-inducible transcription factors (HIFs), which up-regulate the expression of enzymes, transporters, and growth factors. It is unknown whether the expression of genes coding for ion channels is also influenced by hypoxia. We report here that the (cid:1) 1H gene of T-type volt- age-gated calcium channels is highly induced by lower-ing oxygen tension in PC12 cells. Accumulation of (cid:1) 1H mRNA in response to hypoxia is time- and dose-depend-ent and paralleled by an increase in the density of T-type calcium channel current recorded in patch clamped cells. HIF appears to be involved in the response to hypoxia, since cobalt chloride, desferrioxamine, and dimethyloxalylglycine, compounds that mimic HIF-regulated gene expression, replicate the hypoxic effect. Moreover, functional inhibition of HIF-2 (cid:1) protein accumulation using antisense HIF-2 (cid:1) oligonucleotides reverses the effect of hypoxia on T-type Ca 2 (cid:2) channel expression. Importantly, regulation by oxygen tension is specific for T-type calcium channels, since it is not observed with the L-, N-, and P/Q-channel types. These findings show for the first time that hypoxia

Maintaining optimal oxygen homeostasis is of paramount importance for cells. Reductions of oxygen supply trigger cell adaptive responses that minimize the deleterious effects of hypoxia. Cellular responses to hypoxia can be acute, occurring over a time scale of seconds or minutes, or chronic, with time courses of hours to days (1)(2)(3)(4)(5). The major effectors of the acute cellular responses to hypoxia are oxygen-sensitive ion channels. These channels mediate the fast adaptive changes in cell excitability, contractility, and secretory activity that occur in response to low ambient oxygen tension (PO 2 ) (1,5). On the other hand, chronic cellular responses to hypoxia, studied in great detail in the past few years, are mediated by ubiquitously expressed hypoxia-inducible transcription factors (HIF-1␣ 1 and isoforms). Stabilization and transcriptional activity of HIF depend on oxygen-regulated hydroxylases (6 -8). Hypoxia-inducible factors regulate the expression of a wide repertoire of oxygen-sensitive genes with roles in diverse cellular functions such as angiogenesis, red blood cell production, glucose and energy metabolism, apoptosis, and cell proliferation (1)(2)(3)(4)(5).
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 PO 2 is poorly known. There are previous reports showing that prolonged hypoxia down-regulates various voltage-gated K ϩ (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 Ca 2ϩ channel genes. These channels are the major pathway for regulated influx of Ca 2ϩ 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, Ca 2ϩ entry through voltage-gated Ca 2ϩ channels is necessary for the acute response to hypoxia of neurosecretory cells (1). There are two major classes of voltage-dependent Ca 2ϩ channels: low voltageactivated 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 G 1 /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 Ca 2ϩ 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 Ca 2ϩ influx, and catecholamine secretion (20 -22). 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 Ca 2ϩ 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 PO 2 -dependent up-regulation is specific for the T-type Ca 2ϩ channel, since it is barely observed with any of the HVA Ca 2ϩ channels expressed in PC12 cells. These findings define a new role for the T-type Ca 2ϩ channels as regulators of cell excitability and Ca 2ϩ influx during cellular adaptive responses to prolonged hypoxia.

EXPERIMENTAL PROCEDURES
Cell Culture and Treatments-PC12 cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 10% horse serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Cells were routinely cultured in 90% air, 10% CO 2 (normoxic conditions) at 37°C and subcultured every 5 days at 1:5 dilution. For hypoxic treatments, cells at 70 -80% confluence were either exposed to continued normoxia or placed in a hypoxic incubator (Galaxy oxygen control incubator; RS Biotech) that maintained a constant environment (10% CO 2 and either 12, 6, or 3% O 2 balanced with N 2 ) for variable periods of time. In some experiments, cells were switched to Dulbecco's modified Eagle's medium formulated in the absence of calcium (Invitrogen) and with 1 mM EGTA added. This Ca 2ϩ -free medium had to be supplemented with 0.11 mg/ml sodium pyruvate and 4 mM L-glutamine to match the composition of the control medium. Cobalt chloride and desferrioxamine mesylate were obtained from Sigma. Dymethyloxalylglycine was synthesized in facilities of the University of Seville. For electrophysiological recordings, cells were plated on poly-L-lysinecoated glass coverslips prior to the hypoxic treatments.
Reverse Transcription and Polymerase Chain Reaction-Total 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 2-4 g of total RNA using the Superscript TM 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 l of cDNA. The primers used were as follows: rat ␣ 1H , forward (5Ј-GGCGTGGTGGTGGAGAACTT-3Ј) and reverse (5Ј-GATGATGGTGGGATTGAT-3Ј) (GenBank TM accession number AF290213); rat ␣ 1G , forward (5Ј-ACCTGCCTGACACTCTGCA-G-3Ј) and reverse (5Ј-GCTGGCCTCAGCGCAGTCGG-3Ј) (GenBank TM accession number AF290212); and rat ␣ 1I , forward (5Ј-GAGTTAGACA-AGCTCCCAGA-3Ј) and reverse (5Ј-CAGTTGAGGAAGATAAAGGC-3Ј) (GenBank TM accession number AF290214). The PCR products were subcloned in pGemT or pBluescript vectors, and their identities were confirmed by complete DNA sequencing.
Northern Blot Analysis-mRNA was isolated from total RNA with NucleoTrap mRNA minikit (Macherey-Nagel) following manufacturer's instructions. Equal amounts (5 g) 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 [ 32 P]dCTP and Rediprime TM II random prime labeling system (Amersham Biosciences). The probes were subsequently purified with Microspin TM 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 2ϫ SSC, 0.1% SDS at 55°C for 15 min followed by three 5-min washes with 0.5ϫ 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 Ϫ80°C with intensifying screens. Rat cDNA fragments corresponding to nucleotides 4719 -5189 of the ␣ 1H subunit and nucleotides 3568 -4426 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 PCR-Total RNA was isolated from cells after the different treatments, and 4 g 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 l containing 1 or 2 l 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 5Ј-ACTTGGCCATCGTCCTCCT-A-3Ј and reverse 5Ј-GCGGCGTTCATCTCAATCTC-3Ј) were generated to the rat ␣ 1H subunit of T-type Ca 2ϩ channels and used to amplify a 64-base pair fragment. For the ␣-subunits of the HVA Ca 2ϩ channels, primers were as follows: forward (5Ј-TCATCTTCAGCCCAAACAACA-G-3Ј) and reverse (5Ј-TTGGTGAAGATCGTGTCATTGAC-3Ј) for the ␣ 1C subunit (GenBank TM accession number M67516); forward 5Ј-AATGC-CCTGCTCCAGAAAGA-3Ј and reverse 5Ј-CAGACGCTGCCCTAGGTA-AGG-3Ј for the ␣ 1B subunit (GenBank TM accession number M92905); and forward 5Ј-GATGGCTCAAGAAAGCAGCAT-3Ј and reverse 5Ј-G-GCTCCAGGTACCAGTCTTCTG-3Ј for the ␣ 1A subunit (GenBank TM accession number M64373). Melting curve analysis showed a single sharp peak with the expected T m for all samples.
5Ј-Flanking Sequences Analysis-The 5Ј 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 (GenBank TM accession number AF290213) is located in sequence RNOR01500654 on chromosome 10. The mouse ␣ 1H gene (GenBank TM accession number NM_021415) is located in sequence 17.24000001-25000000 on chromosome 17. The human ␣ 1H gene (Gen-Bank TM accession number NM_021098) is located in sequence AC120498.2.1.195680 on chromosome 16.
Electrophysiological Recordings-Macroscopic Ca 2ϩ currents were recorded using the whole cell configuration of the patch clamp technique as adapted to our laboratory (26,27). Patch electrodes (2-3 megaohms) were pulled from hematocrit capillary glass (Hirschmann Laborgerate; 1.5-1.6-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 Ϫ80 mV. Data were filtered at 10 kHz, digitized at a sampling interval of 20 s 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, 22-24°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 BaCl 2 , 1 mM CaCl 2 , 10 mM HEPES, and 10 mM glucose. pH was adjusted with HCl to 7.4, and osmolality was 300 mOsM/kg.
Statistical Analysis-Data were analyzed using Student's t test for unpaired observations with the SigmaPlot program (Jandel). Values are given as mean Ϯ S.E. p values less than 0.05 were considered as statistically significant.

Identification of T-type Ca 2ϩ Channels in PC12 Cells-PC12
are chromaffin-like cells that express HVA Ca 2ϩ channels, which have been identified by biophysical and molecular approaches as L-, N-, and P/Q-subtypes (28,29). To our knowledge, T-type Ca 2ϩ channels have not previously been reported in quiescent PC12 cells. We first tested for the presence of mRNAs coding for T-type Ca 2ϩ channel genes by performing reverse transcriptase-PCR experiments using oligonucleotides specific for each of the three previously identified T-type Ca 2ϩ channel isoforms (␣ 1G , ␣ 1H , and ␣ 1I ) (30). Fig. 1A shows that PCR products of the expected sizes were obtained for the ␣ 1H and ␣ 1G genes but not for the ␣ 1I , which was, however, readily amplified from rat brain RNA. The specificity of the amplification products was further confirmed by complete DNA sequencing. The PCR product for the ␣ 1H gene was consistently more abundant than for the ␣ 1G , suggesting the existence of different levels of expression of the two channel isoforms. Northern blot analyses were then performed to confirm the above results. As shown in Fig. 1B, a probe specific for the ␣ 1H subunit hybridized to a single ϳ8.5-kb transcript in PC12 cells, which was also detected in rat brain as previously reported (30). A second less abundant band of ϳ10 kb, detected in rat brain, was not present in PC12 cells. In contrast to the ␣ 1H subunit, no transcript was detected when a probe specific for the ␣ 1G gene was used, although, as shown in previous reports (31), two mRNAs of ϳ8.5 and ϳ10 kb were observed in rat brain. The absence of signal of ␣ 1G mRNA in Northern blots and the PCR data suggest the existence of a very low level of expression of the ␣ 1G as compared with the ␣ 1H subunit. Altogether, these results reveal that PC12 cells express genes that encode for the ␣ 1H and ␣ 1G subunits of T-type Ca 2ϩ channels, ␣ 1H mRNA being the most abundant.
The presence of functional T-type Ca 2ϩ channels in PC12 cells was confirmed by analysis of the total Ca 2ϩ currents recorded with the whole cell configuration of the patch clamp technique. Fig. 1C shows a representative example of Ca 2ϩ current recorded in a PC12 cell during a depolarizing pulse to ϩ20 mV followed by repolarization to Ϫ70 mV. Consistent with other analyses on Ca 2ϩ 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 Ϫ70 mV. The apparent activation threshold of the whole cell Ca 2ϩ current in PC12 cells was observed at about Ϫ50 mV, and maximal current amplitude was observed at ϩ20 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 Ca 2ϩ channels, which are known to close about ϳ10 -20 times more slowly than HVA channels (12,27,(32)(33)(34). 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 Ϫ70 mV, average fast and slow values were 0.11 Ϯ 0.005 and 1.34 Ϯ 0.09 ms, respectively (n ϭ 45 cells). These data are compatible with the existence of two populations of Ca 2ϩ 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 Ca 2ϩ currents in PC12 cells was further confirmed by their rapid inactivation, another distinct feature of T-type Ca 2ϩ channels (27,32,35,36). Fig. 1E shows representative examples of the deactivating currents recorded in PC12 cells upon repolarization of the cell to Ϫ70 mV after a depolarizing pulse to ϩ20 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, Ca 2ϩ currents in PC12 cells possess a slowly deactivating and fast inactivating component typical of T-type Ca 2ϩ channels. The molecular and the

FIG. 1. Expression of T-type Ca 2؉ channels in PC12 cells. A,
agarose gel separation of the reverse transcriptase-PCR products obtained from total RNA from PC12 cells or rat brain using primers specific for each ␣-subunit. Molecular size of the standards (in kb) is shown on the left. B, Northern blot analysis of the ␣ 1H and ␣ 1G mRNAs obtained from PC12 cells and rat brain tissue. The level of the cyclophilin (CP) mRNA was used to normalize the amount of RNA in each lane. Note that ␣ 1G did not appear in PC12 cells, although the amount of mRNA loaded in this lane was higher than in the lane with brain mRNA. C, macroscopic calcium current recorded in normoxic conditions from a PC12 cell subjected to whole cell patch clamp during a depolarization to ϩ20 mV from a holding potential of Ϫ80 mV. The decay of the tail current generated upon repolarization to Ϫ70 mV (vertical arrow) reflects the closing time course of the channels open during the pulse. Note that although the tail current is fast, it has a clear slow component indicated by the small arrows. D, single (top) and double (bottom) exponential functions fitted to the Ca 2ϩ tail current recorded from a representative PC12 cell. In the double exponential fit, the fast and slow time constant values were 0.09 and 1.79 ms, respectively. E, superposition of normalized tail currents generated upon return to Ϫ70 mV after short (10-ms) or long (50-ms) depolarizing pulses. Reduction in the amplitude of the slow component of the tail current after the 50-ms pulse indicates inactivation of the T-type channels during the long lasting depolarization.
electrophysiological data indicate that ␣ 1H is the major T-type Ca 2ϩ channel subunit functionally expressed in PC12 cells.
Selective Induction of T-type Ca 2ϩ Channel Gene Expression by Hypoxia-Northern 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 PO 2 (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 hypoxiaresponsive 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: ϳ5-fold accumulation of the ␣ 1H mRNA after 6 h of exposure to low PO 2 and a maximal effect at 12-24 h (ϳ8-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 (ϳ1.5-and ϳ2.2-fold induction, respectively) and reaching a maximal induction (ϳ5-8-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 Ca 2ϩ channel gene is up-regulated by hypoxia in a timeand dose-dependent manner.
The data described above prompted us to determine whether up-regulation of gene expression by low PO 2 was selective for the T-type Ca 2ϩ channel or whether hypoxia also altered the expression of other Ca 2ϩ 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 Ca 2ϩ channels, whereas it is a powerful and selective stimulus for T-type Ca 2ϩ channel expression.
Hypoxia Increases the Density of Functional T-type Ca 2ϩ Channels-Electrophysiological experiments were performed to investigate the effect of hypoxia on the level of functional T-type Ca 2ϩ channels in PC12 cells. For these experiments, PC12 cells were either exposed to 3% oxygen for 18 -20 h or incubated under normoxic conditions. Peak inward current density measured at the end of 10-ms pulses to ϩ20 mV showed no differences in the amount of Ca 2ϩ current generated in normoxia (8.6 Ϯ 0.6 pA/pF, n ϭ 45) and hypoxia (8.0 Ϯ 0.6 pA/pF, n ϭ 42). The average I/V relationship was also slightly altered by low PO 2 with only an increase in the density of inward currents in the range between Ϫ50 and Ϫ10 mV (Fig.  4A). Despite the smallness of Ca 2ϩ currents at these voltages, this last observation suggested that the density of functional T-type Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ 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 Ϫ70 mV remained unchanged ( fast ϭ 0.12 Ϯ 0.01 and slow ϭ 1.37 Ϯ 0.06 ms, n ϭ 42 cells; p Ͼ 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 M nickel to the external solution (Fig. 5, E and F), thus further indicating that this component of the Ca 2ϩ current represented the activity of T-type Ca 2ϩ channels (37).
Induction of T-type Ca 2ϩ channel expression by chronic hypoxia is further illustrated in Fig. 6. The average current density due to T-type Ca 2ϩ channels measured during repolarizations from ϩ20 to Ϫ70 mV (3.2 Ϯ 0.47 pA/pF, n ϭ 45 cells) increased almost 2.5 times upon exposure of the cells to hypoxia (7.4 Ϯ 1.4 pA/pF, n ϭ 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 Ca 2ϩ 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 Ttype Ca 2ϩ channels in PC12 cells.
T-type Ca 2ϩ Channel Induction Depends on HIF Activation-It 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 h. E, superposition of calcium current recordings obtained from a PC12 cell after being exposed to chronic hypoxia (3% oxygen for 20 h) and bathed in the control external solution (c and r, control and recovery traces) or in the presence of 50 M nickel chloride added (Ni). Note that nickel reversibly blocks the slow component of the tail current. F, selective reduction by nickel (50 M) of the slow component of the calcium tail currents in PC12 cells (n ϭ 10) after being exposed to hypoxia (3% oxygen) for 20 h. Current density (pA/pF) is indicated in the ordinate. There was no statistically significant difference between the paired I fast /C values. Statistical significance of the differences between the paired I slow /C values was p Ͻ 0.05.
inhibitors of the prolyl hydroxylases that regulate the proteasomal degradation of HIF isoforms (HIF-1␣ and HIF-2␣) (4-7, 38, 39). As shown in Fig. 7, A and B, Northern blot and real time PCR experiments revealed that treatment of PC12 cells with CoCl 2 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 (1-5), HIF is involved in the hypoxic up-regulation of T-type Ca 2ϩ channel expression.
To determine whether HIF is indeed functionally involved in the hypoxic up-regulation of T-type Ca 2ϩ 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 antisense 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 Ca 2ϩ channel suggested by these experiments is further supported by the presence of hypoxia-responsive elements in the 5Ј-flanking region of the ␣ 1H gene. We identified numerous sequences compatible with hypoxia-responsive elements (44, 46 -49) in a region of ϳ1300 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.
Dependence on Ca 2ϩ of the Regulation of T-type Ca 2ϩ Channel Gene Expression by Hypoxia-One of the earliest known responses to hypoxia in PC12 cells is membrane depolarization and increase of intracellular Ca 2ϩ levels (20). Previous reports have shown that increase of intracellular free Ca 2ϩ is required FIG. 6. Increase of the density of T-type Ca 2؉ channels in chronic hypoxia. A, current density (expressed in pA/pF) of the slowly deactivating component of the calcium currents recorded at Ϫ70 mV in normoxia (21% oxygen, 3.2 Ϯ 0.47 pA/pF, n ϭ 45 cells) and hypoxia (3% oxygen, 7.4 Ϯ 1.4 pA/pF, n ϭ 42 cells). Statistical significance between the two groups was p Ͻ 0.01. B, current density (expressed in pA/pF) of the fast deactivating components of the calcium currents recorded at Ϫ70 mV in normoxia (21% oxygen, 30.4 Ϯ 2.79 pA/pF, n ϭ 45 cells) and hypoxia (3% oxygen, 21.3 Ϯ 2.27 pA/pF, n ϭ 42 cells). Statistical significance between the two groups was p ϭ 0.04. C, ratio of current densities (slow/fast) calculated for each cell in the two experimental conditions. Statistical significance between the two groups was p Ͻ 0.01. 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 Ca 2ϩ -dependent, PC12 cells were incubated in Ca 2ϩ -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 Ca 2ϩ . Thus, an increase in intracellular free Ca 2ϩ does not seem to be required for induction of ␣ 1H gene expression by hypoxia in PC12 cells. In contrast, it seems that maintained Ca 2ϩ influx might even down-regulate ␣ 1H expression. 2

DISCUSSION
For the last decade, research on cellular oxygen homeostasis has rapidly progressed, due, on one hand, to the characterization of the HIF-dependent pathway that regulates the expression of enzymes and growth factors in response to chronic deficiency of O 2 and, on the other, to the identification of O 2 -sensitive ion channels as mediators of the acute cardiocirculatory and respiratory reflexes evoked by hypoxia (1,2,5,51). There is, however, almost no knowledge of the long lasting adaptive changes of ion channel gene expression in prolonged hypoxia. Among all voltage-sensitive ion channels, the T-type Ca 2ϩ channels are of special interest, because they regulate cellular excitability, which changes during acute and protracted hypoxia, and appear to be also involved in cellular proliferation, which is tightly constrained by the need of precise oxygen homeostasis. We report here that T-type Ca 2ϩ channels are expressed in PC12 cells. More importantly, we show both at the molecular and the functional levels that hypoxia greatly induces the expression of the ␣ 1H subunit of T-type Ca 2ϩ channels through activation of the HIF-dependent pathway.
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 Ca 2ϩ channels, PC12 cells also express a smaller population of slowly deactivating and fast inactivating T-type Ca 2ϩ channels. Significantly, the estimated deactivation time constant of the native T-type Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ channels from the fast deactivating HVA channels in previous electrophysiological studies on PC12 cells. Likewise, T-type Ca 2ϩ 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 Ca 2ϩ 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,(32)(33)(34)37). Induction of the ␣ 1H subunit by hypoxia was time-and dose-dependent, reaching maximal levels (about 5-8-fold) after 12-24 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 Ca 2ϩ channel genes expressed in PC12 cells. Neither significant accumulation of the ␣ 1C (L-type), ␣ 1B (N-type), and ␣ 1A (P/Qtype) mRNAs nor significant increase of the fast deactivating Ca 2ϩ 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 Ca 2ϩ channels, there is a previous report showing that chronic hypoxia increases the amplitude of Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ channel expression by low PO 2 has the same general features (O 2 levels, time course, etc.) of the classical hypoxia-inducible genes such as erythropoietin or vascular endothelial growth factor (1)(2)(3)(4)(5). Although there exists the possibility that hypoxia-induced up-regulation of the ␣ 1H Ca 2ϩ 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 Ca 2ϩ channel gene expression by low PO 2 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-, Fe 2ϩ -, and oxoglutarate-dependent dioxygenase that under normoxic conditions hydroxylates specific proline and asparagine residues in HIF prior its degradation (6 -8). 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 Ca 2ϩ channel expression. This experimental observation is in fair agreement with the existence in the 5Ј-flanking region of the ␣ 1H gene of several putative HIF consensus DNA binding sites (46 -49). We have also investigated whether Ca 2ϩ is critical for the hypoxiainduced regulation of ␣ 1H Ca 2ϩ channel expression. Interestingly, the increase in ␣ 1H mRNA induced by hypoxia was completely unaffected by the removal of extracellular Ca 2ϩ . This finding contrasts with the observed Ca 2ϩ dependence of the hypoxic induction of some genes in PC12 cells (24,50). However, recent data demonstrate that intracellular Ca 2ϩ 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 ϩ 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 (60 -72 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 Ca 2ϩ channels, because Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ and enhances the secretory response of PC12 cells to acute hypoxia (21). Chronic intermittent exposures to low PO 2 have been shown to increase the excitability of carotid body and sympathoadrenal tissues (61). Besides the possible role of Ttype 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 Ca 2ϩ entry through T-type Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ channels in cell growth and tumor biology.