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Multiple transcripts of Ca²⁺ channel ₁-subunits and a novel spliced variant of the _1C-subunit in rat ductus arteriosus

Departments of ¹Pediatrics, ²Physiology, and ³Neuroanatomy, Yokohama City University, Yokohama; ⁴Consolidated Research Institute for Advanced Science and Medical Care, Waseda University; ⁵Department of Pharmacology, School of Medicine, Faculty of Medicine, Toho University, Tokyo; ⁶Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan; and ⁷Cardiovascular Research Institute, Departments of Cell Biology and Molecular Medicine and Medicine (Cardiology), New Jersey Medical School, Newark, New Jersey

Submitted 3 February 2004 ; accepted in final form 27 October 2005

	ABSTRACT

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Voltage-dependent Ca²⁺ channels (VDCCs), which consist of multiple subtypes, regulate vascular tone in developing arterial smooth muscle, including the ductus arteriosus (DA). First, we examined the expression of VDCC subunits in the Wistar rat DA during development. Among ₁-subunits, _1C and _1G were the most predominant isoforms. Maternal administration of vitamin A significantly increased _1C- and _1G-transcripts. Second, we examined the effect of VDCC subunits on proliferation of DA smooth muscle cells. We found that 1 µM nitrendipine (an L-type Ca²⁺ channel blocker) and kurtoxin (a T-type Ca²⁺ channel blocker) significantly decreased [³H]thymidine incorporation and that 3 µM efonidipine (an L- and T-type Ca²⁺ channel blocker) further decreased [³H]thymidine incorporation, suggesting that L- and T-type Ca²⁺ channels are involved in smooth muscle cell proliferation in the DA. Third, we found that a novel alternatively spliced variant of the _1C-isoform was highly expressed in the neointimal cushion of the DA, where proliferating and migrating smooth muscle cells are abundant. The basic channel properties of the spliced variant did not differ from those of the conventional _1C-subunit. We conclude that multiple VDCC subunits were identified in the DA, and, in particular, _1C- and _1G-subunits were predominant in the DA. A novel spliced variant of the _1C-subunit gene may play a distinct role in neointimal cushion formation in the DA.

alternative spliced; development; gene expression; fetal circulation

VDCCs are classified, according to their distinct electrophysiological and pharmacological properties, into low (T-type) and high (L-, N-, P-, Q-, and R-type) VDCCs (20, 39). VDCCs consist of different combinations of ₁-subunits and auxiliary subunits. The ₁-subunit forms the ion-conducting pore, the voltage sensor, and the interaction sites for Ca²⁺ channel blockers and activators (15). Therefore, ₁-subunits principally determine the channel character of VDCCs. Ten ₁-subunit isoforms have been identified. Four ₂-subunit complexes and four -subunits, which modulate the trafficking and the biophysical channel properties of ₁-subunits (1), have been identified (3). Although some studies have investigated the role of VDCCs in the DA (28, 37), characterization of VDCCs, including the composition of each subunit, the developmental change in their expression, and their physiological roles, remains poorly understood.

In addition to their role in determining contractile state, a growing body of evidence has demonstrated that VDCCs play an important role in regulating differentiation and remodeling of vascular smooth muscle cells (SMCs) (14, 17, 41). The DA dramatically changes its morphology during development. Intimal cushion formation during development is a characteristic feature of vascular remodeling of the DA (10, 30). Intimal cushion formation involves many cellular processes, including an increase in SMC migration and proliferation, production of hyaluronic acid under the endothelial layer, and impairment of elastin fiber assembly. The role of VDCCs in vascular remodeling of the DA has not been investigated.

In the present study, we identified multiple VDCC subunits in the DA by semiquantitative and quantitative RT-PCR and immunodetection. In particular, _1C- and _1G-subunits were predominant in the DA. Furthermore, we will demonstrate the identification of a novel spliced variant of the _1C-subunit gene that may play a role in neointimal cushion formation of the DA.

	MATERIALS AND METHODS

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Animals. All animals were cared for in compliance with the guiding principles of the American Physiological Society. The experiments were approved by the Ethical Committee of Animal Experiments of Yokohama City University School of Medicine.

Maternal vitamin A administration. Maturation of the fetal DA was accelerated by maternal administration of vitamin A, as described previously (25, 42). Briefly, vitamin A [retinyl palmitate, 33 mg, which is equivalent to 18 mg of retinol (Eisai, Tokyo, Japan)] was diluted in polyoxyethylene castor oil and injected intramuscularly into pregnant Wistar rats daily from day 17 of gestation in a dose of 1 mg (3,000 IU)/kg body wt.

Tissue collection and preparation. For developmental studies, we used pooled tissues obtained from Wistar rat embryos on embryonic days 19 (e19, n = 60) and 21 (e21, n = 90) and from neonates on the day of birth (day 0, n = 60). After excision, tissues were immediately frozen in liquid nitrogen and stored at –80°C.

Semiquantitative and quantitative RT-PCR. Total RNA was isolated from the tissues using TRIzol, as recommended by the manufacturer (Invitrogen, La Jolla, CA). Genomic DNA was digested by DNase I before RT reaction. After annealing to random hexamer, 2 µM total RNA was reverse transcribed to cDNA by SuperScript II RT (Invitrogen). For semiquantitative RT-PCR analyses of VDCC subunits, the primers for PCR amplification were designed on the basis of the rat nucleotide sequences of VDCCs (Table 1) to amplify cDNA between two exons, except _1S-subunit primers. The PCR cycle consisted of denaturation at 94°C for 30 s, annealing at each temperature for 30 s, and elongation at 72°C for 30 s; 35–40 forty cycles were performed. Amplification products were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining. A fragment of GAPDH was amplified as internal control. For quantitative RT-PCR analysis, sequences for PCR primers are listed in Table 2. The spliced variant of the _1C-subunit and nonspliced isoform were detected simultaneously by Ca_v1.2 (_1C)-4 primer. Amplification and detection were performed as described previously (40). Each template was tested at least three times to confirm the reproducibility of the assays. The abundance of each gene was determined relative to GAPDH using TaqMan rodent GAPDH control reagents kits (Applied Biosystems, Foster City, CA). For each RT-PCR experiment, we included RT negative control and confirmed no amplification in each reaction.

Generation of polyclonal antibody against spliced variant of _1C-subunit. We generated a polyclonal antibody against spliced variant of rat _1C-subunit using a keyhole limpet hemocyanin-conjugated synthetic peptide for immunization. Antigens for spliced variant of anti-rat _1C-subunit antibody were derived from the I-II cytoplasmic loop region spanning amino acids 59–71 of the spliced variant of rat _1C-subunit (RGAPAGLHDQKKG+C). A male Japanese White rabbit was immunized four times every 2 wk. Serum was collected, and polyclonal antibody was affinity purified.

Immunoblotting. The membrane fraction was prepared and immunoblotting was performed as described previously (23). Briefly, tissues from rat (DA, aorta, atria, left ventricle, and lung) were homogenized in an ice-cold buffer [in mM: 50 Tris (pH 8.0), 1 EDTA, 1 EGTA, 1 dithiothreitol, and 200 sucrose] and protease inhibitors (Complete Mini, Roche, Tokyo, Japan). The polyclonal antibody specific for _1C-, _1D-, and _1G-subunits (Chemicon, Temecula, CA) or spliced variant of _1C-subunit at 5 µg/ml was used to examine 20-µg membrane fractions from rat tissues.

Immunohistochemistry. For immunoperoxidase demonstration of VDCCs in the DA, paraffin-embedded blocks containing DA tissues were cut into 4-µm-thick sections and placed on 3-aminopropyltriethoxysilane-coated glass slides. To determine the boundary line of intimal cushion formation, tissue sections were stained with Elastica van Gieson as recommended by the manufacturer (Muto Pure Chemicals). The specimens were deparaffinized, rehydrated, and incubated for 5 min in peroxidase-blocking reagent (DAKO Laboratories) to inactivate endogenous peroxidases. Slides were incubated with each primary antibody of splicing variant of _1C-, _1D-, _1G-, and _1C-subunits (1:200 dilution) at room temperature for 30 min. After they were washed with 0.1 M PBS for 5 min, the slides were incubated for 30 min in biotinylated rabbit anti-goat IgG (Vector, Burlingame, CA). Then the slides were washed with 0.1 M PBS for 5 min, incubated for 30 min in avidin-biotin-horseradish peroxidase complex (Vector), and washed again with 0.1 M PBS for 5 min. The peroxidase reactivity was demonstrated with 3,3’-diaminobenzidine (Sigma, St. Louis, MO) and 0.3% H₂O₂ for 5 min. The specificity of staining was examined by omission of the primary antibodies. The slides were counterstained with Mayer’s hematoxylin.

Primary culture of rat DA SMCs. Vascular SMCs in primary culture were obtained from the DA of Wistar rat embryos at e21. The tissues were minced and transferred to a 1.5-ml centrifuge tube that contained 800 µl of collagenase-dispase enzyme mixture [1.5 mg/ml collagenase-dispase (Roche), 0.5 mg/ml elastase type II-A (Sigma Immunochemicals, St. Louis, MO), 1 mg/ml trypsin inhibitor type I-S (Sigma), and 2 mg/ml bovine serum albumin fraction V (Sigma) in Hanks’ balanced salt solution (Sigma)]. The digestion was carried out at 37°C for 15–20 min. Then cell suspensions were centrifuged, and the medium was changed to the collagenase II enzyme mixture [1 mg/ml collagenase II (Worthington), 0.3 mg/ml trypsin inhibitor type I-S, and 2 mg/ml bovine serum albumin fraction V in Hanks’ balanced salt solution]. After 12 min of incubation at 37°C, cell suspensions were transferred to growth medium in 35-mm poly-L-lysine (Sigma)-coated dishes in a moist tissue culture incubator at 37°C in 5% CO₂-95% ambient mixed air. The growth medium contained DMEM with 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). The confluent cells were used at passages 4–6. We confirmed that >99% of cells were positive for -smooth muscle actin and showed the typical "hill-and-valley" morphology.

Cell proliferation assays. [³H]thymidine incorporation was used to measure cell proliferation in DA SMCs. The SMCs were reseeded into a 24-well culture plate at an initial density of 1 x 10⁵ cells per well for 24 h before serum starvation with DMEM containing 0.5% FCS. Cells were then incubated with or without nitrendipine (1 µM), kurtoxin (1 µM), and efonidipine (3 µM) for 16 h in the starvation medium before addition of 1 µCi of [methyl-³H]thymidine (specific activity 5 Ci/mM; Amersham International, Bucks, UK) for 4 h at 37°C. After fixation with 1.0 ml of 10% trichloroacetic acid, the cells were solubilized with 0.5 ml of 0.5 M NaOH and then neutralized with 0.25 ml of 1 N HCl. A liquid scintillation counter was used to measure [³H]thymidine incorporation. Data obtained from triplicate wells were averaged.

Generation of expression construct for spliced variant of rat _1C-subunit. The 220-bp fragment containing a 75-bp insertion of the spliced variant of rat VDCC _1C-subunit was extracted from the 453-bp PCR fragment using Xho I and Sph I restriction enzymes. Then the 220-bp fragment was introduced into the Xho I/Sph I site of the pcDNA3.1(+)-based expression construct of the rat brain 1C subunit (rbCII; kindly supplied by Dr. T. P. Snutch) (35). The sequence of the expression construct was confirmed by direct sequencing analysis.

Electrophysiological recordings. Ca_V1.2 (rbCII; GenBank accession no. M67515) or its mutant was transiently expressed in BHK6 cells, which stably express ₁- and ₂-subunits. Transfection was carried out with a transfection reagent (FuGene 6, Roche), as previously described (43).

Electrophysiological recordings were performed in the whole cell patch-clamp configuration using a patch/whole cell-clamp amplifier (Axopatch 200B, Axon Instruments) and an analog-to-digital converter (Digidata 1200, Axon Instruments) (43). Data acquisition was performed with pCLAMP7 software (Axon Instruments). Signals were filtered at 5 kHz. Capacitative currents were electrically compensated. The P/4 protocol (pCLAMP7) was used for leak subtraction. Ca²⁺ currents and Ba²⁺ currents (I_Ba) through Ca_V1.2 (rbCII) Ca²⁺ channels expressed in BHK6 cells were measured as previously described (27). The external solution contained (in mM) 137 NaCl, 5.4 KCl, 1 MgCl₂, 10 HEPES, and 10 glucose, with 2 CaCl₂ or BaCl₂ as a charge carrier; pH was adjusted to 7.4 with NaOH at room temperature. The resistance of the patch electrode was 2–2.5 M when it was filled with the pipette solution containing (in mM) 120 CsMeSO₄, 20 TEA-Cl, 14 EGTA, 5 Mg-ATP, 5 Na₂ creatine phosphate, 0.2 GTP, and 10 HEPES, with pH adjusted to 7.3 with CsOH at room temperature. All experiments were carried out at room temperature.

The half-activation potential (V_h-act) was estimated by fitting the current-voltage (I-V) relations (curves) to the following equation by an interactive nonlinear regression fitting procedure

where V_m is membrane potential, V_rev is reversal potential, k is slope factor, and G_max is maximum conductance.

Half-inactivation voltage (V_h-inact) was estimated by fitting the steady-state inactivation curves to the following equation

where I_max and I_min are maximum and minimum plateau currents, respectively, and k is slope factor.

Statistical analysis. Values are means ± SE. Student’s unpaired t-tests and unpaired ANOVA followed by the Student-Newman-Keuls test were used for statistical analysis. P < 0.05 was considered statistically significant.

	RESULTS

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Multiple transcripts of VDCC ₁-, ₂-, and -subunits in rat DA. Semiquantitative RT-PCR analyses revealed that, among voltage-dependent L-type Ca²⁺ channel subunits, _1C- and _1D-subunit mRNAs were expressed in the DA and the aorta, whereas neither _1F- nor _1S-subunit transcript was detected (Fig. 1A). The _1C-subunit transcripts were amplified as two bands, 378 bp (the expected size) and 453 bp, in the RT-PCR products. The 378-bp band was confirmed as the reported VDCC _1C-subunit, and the 453-bp band was identified as a novel spliced variant of the rat VDCC _1C-subunit by sequencing analysis. Another spliced variant of _1C-subunit in the human, which displayed oxygen-sensitive opening of the channel, was recently identified (11). However, we could not detect this spliced variant in DA, aorta, and genomic DNA in the rat by RT-PCR using Ca_v1.2 (_1C)-2 primers, although we could detect its expression in human right ventricle (data not shown). The expression of VDCC _1A (P/Q-type)-, _1B (N-type)-, and _1E (R-type)-subunits was not detected in the DA by semiquantitative RT-PCR (data not shown).

View larger version (55K): [in this window] [in a new window]   Fig. 1. Semiquantitative RT-PCR analyses of Ca2+ channel subunits. A: RT-PCR for L-type Ca2+ channel 1-subunit isoforms in rat ductus arteriosus (DA), aorta, skeletal muscle, and retina. RNA samples from tissues were processed for 35 cycles of PCR using primers directed to the cDNA sequence of VDCC 1S-, 1C-, 1D-, and 1F-isoforms. PCR of the primer alone, without template, resulted in no product (data not shown). Transcripts for 1C- and 1D-subunits were detected in DA and aorta, but no transcripts for 1S- and 1F-subunits were detected in DA and aorta. Transcripts for 1C-subunit were detected as clear bands of expected (378 bp) and longer (453 bp) lengths. Sequence analysis detected insertion of an unreported 75-bp cDNA in the 453-bp band into the 378-bp band. Expression of 1C-subunit mRNA was not altered during development. Expression of 1D-subunit mRNA was decreased in DA from embryonic day 21 (e21) but decreased from day 0 (birth) in aorta. B: RT-PCR for T-type Ca2+ channel 1G-, 1H-, and 1I-subunits in rat DA, aorta, skeletal muscle, and retina. Transcripts for these subunits were present in DA and aorta. Expression level of 1G-subunit mRNA was high from embryonic day 19 (e19) to day 0 in DA and aorta and decreased in adult aorta. Levels of 1H- and 1I-subunit mRNA expression were low in DA and aorta. C: RT-PCR for 2-subunits in rat DA and aorta during development. Transcripts for all 4 2-subunit isoforms were present in both tissues. D: RT-PCR for -subunits in rat DA and aorta during development. Transcripts for 4 -isoforms were present in both tissues. 3-Subunit mRNA was abundant in DA and aorta throughout development.

The transcripts of all four ₂-subunits were detected in the DA and aorta (Fig. 1C). Transcripts of all four -subunits were detected in the DA and aorta (Fig. 1D). Among them, ₃-subunit mRNA was highly expressed by semiquantitative RT-PCR during development in the DA and aorta. Expression of ₃-subunit mRNA was not changed during development. Expression of ₁-subunit mRNA was not detected in adult aorta. Expression ₂- and ₄-subunit mRNA was higher in the fetus than that in neonates on day 0.

VDCC _1C-subunit was a predominant transcript of L-type Ca²⁺ channel subunits in rat DA. Using restriction enzyme analysis as previously reported (29), we determined relative abundance of HVA Ca²⁺ channel mRNA. The digested fragments were detected only in the _1C- and _1D-subunit lanes (Fig. 2), which is consistent with the result from semiquantitative RT-PCR. The values of _1C- and _1D-subunits were obtained when the value into which elements digested by each enzyme was divided by the amount of uncut PCR product. The density of the digested fragment was significantly higher in the _1C- than in the _1D-subunit lane, indicating that the _1C-subunit is the most abundant transcript among L-type Ca²⁺ channel subunits.

View larger version (63K): [in this window] [in a new window]   Fig. 2. Relative abundance of L-type Ca2+ channel 1-subunits in DA at e21. Uncut lane shows a single band corresponding to fragments obtained after RT-PCR; 1C, 1D, and 1S lanes show fragments obtained after restriction digest with enzymes. Digested fragments are indicated by arrows. Digested fragment was detected more in 1C- than in 1D-subunit. No digested fragment was detected in 1S-subunit. Values for 1C- and 1D-subunits were obtained from the value into which elements digested by each enzyme were divided by amount of uncut PCR product using densitometry. The 1C-subunit is the most abundant of the L-type Ca2+ channel subunits. *P < 0.01.

Protein expression of _1C-, _1D-, _1G-, and ₃-subunits in the DA.

View larger version (74K): [in this window] [in a new window]   Fig. 3. A: expression of 1C-, 1D-, 1G-, and 3-subunit protein in rat DA, aorta, and atrium by immunoblotting. Membrane proteins from rat tissues were separated by SDS-PAGE, together with prestained molecular weight markers, and subjected to immunoblot analysis with each subunit-selective antibody. Expression level of 1C-subunit protein was comparable in DA and aorta at e21 and much less in DA than in adult atrium. Expression of 1D-subunit was similarly detected in DA and aorta at e21. Expression of 1G-subunit protein was high in DA and aorta at e21 and undetectable in adult aorta. Expression of 3-subunit was high in DA at e21. B: DA at e21 immunostained with anti-1C, -1D, and -1G. Elastica stain identified the boundary of intimal cushion formation. High-magnification (x4) image of the region of intimal cushion formation is shown at bottom. Strong immunoreaction of 1C-subunit and mild immunoreaction of 1D-subunit were ubiquitously found in DA. Strong 1G-subunit immunoreaction was especially found at the region of intimal thickening (IT) in DA. Scale bar, 100 µm.

Effects of development and maternally administered vitamin A on expression of VDCC ₁-subunit transcripts. Although the expression levels of _1C-subunit mRNA were not changed in the DA during development, a significant decrease in the expression level of _1C-subunit mRNA in the aorta resulted in a higher expression of _1C-subunit mRNA in the DA than in the aorta after e21 (Fig. 4A). Maternally administered vitamin A significantly increased the expression levels of _1C-subunit mRNA at day 0 (P < 0.01; Fig. 4B).

View larger version (31K): [in this window] [in a new window]   Fig. 4. A: developmental changes in expression of 1C-subunit mRNA. Expression level of 1C-subunit mRNA was higher in DA than in aorta (AO) at e21 and day 0. Expression level was not changed in DA during development but was decreased in aorta from e19 to e21. *P < 0.05. NS, not significant. B: effects of maternally administered vitamin A (MVA) on expression of 1C-subunit mRNA in DA. Levels of 1C-subunit mRNA were not changed in DA during development and were significantly increased with vitamin A only at day 0. *P < 0.05; **P < 0.01. C: developmental changes in expression of 1G-subunit mRNA. Transcripts for 1G-subunit were increased during development in DA. Abundance of 1G-subunit mRNA was significantly greater in DA than in aorta at day 0. **P < 0.01; ***P < 0.001. D: effects of maternally administered vitamin A on expression of 1G-subunit mRNA in DA. Expression of 1G-subunit mRNA was upregulated during development and significantly higher than with vitamin A at any developmental stage. **P < 0.01; ***P < 0.001.

Effects of L- and T-type VDCCs on SMC proliferation in DA. We used a specific L-type VDCC blocker (nitrendipine), a specific T-type VDCC blocker (kurtoxin), and an L- and T-type VDCC blocker (efonidipine) to investigate a role for VDCCs in SMC proliferation in the DA. Significant inhibition of [³H]thymidine incorporation was observed in rat DA SMCs treated with 1 µM nitrendipine, 3 µM efonidipine, or 1 µM kurtoxin compared with untreated SMCs (Fig. 5). The inhibition was the strongest in SMCs treated with 3 µM efonidipine, suggesting that the additive inhibitory effect of efonidipine on cell proliferation is due to the blockade of L- and T-type VDCCs in rat DA SMCs.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Effects of VDCC blockers on [3H]thymidine uptake of DA smooth muscle cells. [3H]thymidine incorporation in groups treated with nitrendipine, efonidipine, and kurtoxin was decreased compared with that in control group in 0.1% FCS-containing medium. Treatment of DA smooth muscle cells with efonidipine resulted in additional reduction of [3H]thymidine uptake compared with nitrendipine or kurtoxin treatment. Experiments were performed 3 times independently in duplicate. Significantly different from control: *P < 0.01, **P < 0.0001. Significantly different from nitrendipine (P < 0.01). Significantly different from kurtoxin (P < 0.01).

A novel spliced variant of the _1C-subunit was highly expressed in adult lung and fetal arteries.

View larger version (44K): [in this window] [in a new window]   Fig. 6. A: alignment of nucleotide sequence of rat Ca2+ channel 1C-subunit (GenBank accession no. AF394938) and novel spliced variant (GenBank accession no. AY323810). Spliced variant consists of a 75-bp insertion. B: amino acid sequence of rat Ca2+ channel 1C-subunit. Spliced variant contains a 25-amino acid insertion into the I-II cytoplasmic linker that interacts with the -subunit of 1C. C: comparison of amino acid sequence of novel 1C-subunit alternatively spliced isoform among several species. Sets of conservative or unconservative residues are indicated in bold or light font, respectively. Amino acid sequence is highly conserved among species.

View larger version (62K): [in this window] [in a new window]   Fig. 7. A: expression of splicing variant of 1C-subunit confirmed by semiquantitative RT-PCR and immunoblotting analyses. Band migrating at 453 bp is spliced variant of 1C-subunit; smaller band is nonspliced 1C-isoform. Immunoblotting analysis revealed that spliced variant of 1C-subunit was highly expressed in vascular smooth muscle, including DA, at mRNA and protein levels. B: relative abundance of spliced variant of 1C-subunit mRNA measured by quantitative RT-PCR. Level of spliced variant of 1C-subunit mRNA is highly expressed in DA and aorta in the fetus. C: immunoreaction of splicing variant of 1C-subunit was found in smooth muscle cells of DA at e21. Strong immunoreaction was found in region of intimal thickening. Scale bar, 100 µm. High-magnification (x4) image of region enclosed in rectangle at left is shown at right.

Localization of the spliced variant of the _1C-subunit in the DA at e21 was examined by immunostaining with anti-_1C-subunit splicing variant (Fig. 7C). Strong immunoreaction was found in the region of intimal thickening of the DA (Fig. 7C, right), whereas immunoreaction of the conventional _1C-isoform was ubiquitously expressed in the whole layers of the DA (Fig. 3B).

We examined whether the DA variant exerts any differences in the gating kinetics of the Ca²⁺ channel. Activation and inactivation kinetics of I_Ba were not significantly different between rbCII and the DA variant (Fig. 8A). The expression level at the surface membrane, estimated as the density of I_Ba, did not differ between the two groups, even though the individual cell showed a wide variety of I_Ba density, as is often the case with a transient expression experiment (Fig. 8B). The I-V relations of rbCII and the DA variant were almost superimposable (Fig. 8C). V_h-act of rbCII and the DA variant were –15.4 ± 1.9 mV (n = 9) and –14.5 ± 1.2 mV (n = 7), respectively (not statistically significant). The steady-state inactivation curves could be slightly shifted toward the depolarized direction in the DA variant, but V_h-inact was not significantly different: –33.3 ± 1.0 mV (n = 9) and –31.5 ± 1.7 mV (n = 6), respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 8. A: Ba2+ current (IBa) traces of rat brain 1C subunit (rbCII) and the DA variant. Peak normalized currents were averaged and superimposed. Pulse protocol is indicated above current traces. B: voltage dependence of IBa density of DA variant and rbCII. C: current-voltage (I-V) relations (curve) of rbCII and the DA variant. Amplitude of IBa was normalized to peak value in each recording and averaged. D: steady-state inactivation curves of rbCII and the DA variant. Test pulse to 0 mV for 100 ms was applied after conditioning pulse for 5 s to the respective voltages ranging from –80 to 10 mV in 10-mV steps and for 25 ms at –70 mV. Values are means ± SE.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

We also found that all T-type VDCCs were expressed in the DA. The most dominant isoform among T-type VDCCs in the DA is the _1G-subunit, with an expression level 25–120 times higher in fetal vessels than in adult aorta, which is consistent with previous reports showing that T-type VDCCs are predominantly expressed in the early stages of differentiation of many embryonic and neonatal tissues (2, 9, 12, 21). The expression of _1G-subunit mRNA was significantly upregulated by maternal administration of vitamin A. In the DA, _1G-subunit protein was highly localized in the region of intimal thickening. Although the abundant expression of the _1G-subunit suggests that the _1G-subunit plays an important role in the DA, the physiological role of T-type VDCCs in smooth muscle contraction has been obscure. However, Ca²⁺ influx through the _1H-subunit has been recently identified to be essential for normal relaxation of coronary arteries (4). Therefore, T-type VDCCs may play a similar role in the DA, rather than in oxygen-sensitive contraction (38). Further investigation is necessary to test the possibility.

In addition to the regulation of vascular tone, L- and T-type VDCCs are also known to regulate differentiation (14, 17), proliferation (19, 36, 44), migration (7, 31), and gene expression (41) in vascular SMCs. In the present study, we found that L- and T-type Ca²⁺ channel blockers significantly inhibited [³H]thymidine incorporation in DA SMCs, suggesting that L- and T-type VDCCs promote cell proliferation in the DA. Moreover, we found that BAY K 8644, an L-type VDCC activator, increased DA SMC migration in a dose-dependent manner (unpublished data). A previous study demonstrated that the blockade of T-type, but not L-type, VDCCs prevented neointima formation after vascular injury (32), which shares a molecular mechanism of intimal thickening similar to that of the DA. Our present results, however, indicate that L- and T-type VDCCs are involved in intimal thickening in the DA in different ways.

Previous studies demonstrated that responses of the DA to oxygen and indomethacin, a prostaglandin H synthase inhibitor, are blunted at e19 and are apparent at e21 (24, 25). In this study, the expression level of _1C-subunit mRNA at e19 was similar to that at e21 or day 0. Therefore, the expression level of the _1C-subunit was not considered the cause of the blunted response of the DA to oxygen and indomethacin at e19. One may argue that an oxygen-sensitive signal is activated at near term (e21) to increase the activity of VDCCs. In this sense, vitamin A and/or retinoic acid signaling is a candidate for the activator of oxygen sensitivity, because the retinoic acid response element is strongly expressed in the mouse DA (6), and maternally administered vitamin A accelerated development of the oxygen-sensing mechanism of the rat DA (42). Previous studies have also demonstrated that retinoic acid upregulated _1C-subunit L-type Ca²⁺ channel expression in vascular SMCs (13) and H₉C₂ cardiac myoblast cell lines (22). Although vitamin A upregulated the expression of _1C-subunit mRNA in the DA only at day 0, it would be of great interest that vitamin A and/or retinoic acid signal may enhance the activity of VDCCs in the DA.

We found a novel spliced variant of the _1C-subunit in the rat. The spliced variant contained a 25-amino acid insertion into the I-II cytoplasmic linker. The interaction between the cytoplasmic I-II linker of _1C- and -subunits is known to modulate channel opening (16). During the preparation of this manuscript, Liao et al. (18) reported the same spliced variant of the _1C-subunit. They demonstrated that the spliced variant of the _1C-subunit exhibited a hyperpolarized shift in voltage-dependent activation and the I-V relation in HEK 293 cells. However, we did not find a difference in basic electrophysiological channel properties between the conventional and the spliced variant of _1C-subunits. Although we do not explain an exact reason for the conflicting results between two studies, the discrepancy may be due to the different conditions of the experiments: we used rat cDNA and the ₁-subunit, whereas Liao et al. used human cDNA and the ₂-subunit. Although we did not find a difference in basic channel properties between the conventional and the spliced variant of _1C-subunits, we found the distinct expression pattern of the spliced variant in the DA. The spliced variant was strongly expressed in neointimal thickening of the DA, where SMCs exhibit more proliferating and migrating characters (33, 34). In addition, we found that expression of the spliced variant mRNA was significantly increased in the lung of monocrotaline-treated rats (unpublished data). These results suggest a distinct role for the spliced variant in adaptation to various physiological and/or pathological signals.

In conclusion, multiple VDCC subunits were identified in the DA, and, in particular, _1C- and _1G-subunits were predominant in the DA. The expression of _1C- and _1G-subunit mRNAs was higher in the DA than in the aorta and was significantly upregulated by maternal administration of vitamin A. We found a novel spliced variant of the _1C-subunit gene that may play a specific role in Ca²⁺ entry in the lung and fetal arteries. Our study could be an important first step in identification of the molecular basis of Ca²⁺ channel function in the DA. On the basis of our results, further study would identify the physiological relevance between mRNA levels and Ca²⁺ channel function in the DA.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was partly supported by the Mother and Child Health Foundation (S. Minamisawa), 2005 Strategic Research Project Grant K17014 of Yokohama City University (S. Minamisawa), the Yokohama Foundation for Advancement of Medical Science (U. Yokoyama and T. Akaike), and a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science (S. Adachi-Akahane).

	ACKNOWLEDGMENTS

 
We are grateful to Takayo Musuda, Mayumi Watanabe, and Chikako Usami for excellent technical assistance and animal care. Efonidipine was kindly provided by Nissan Chemical Industries (Saitama, Japan).

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. Minamisawa, Dept. of Physiology, Yokohama City Univ., 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan (e-mail: sminamis{at}med.yokohama-cu.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

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