L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells

^a Franz-Volhard Clinic at the Max-Delbrück Center for Molecular Medicine, Humboldt University of Berlin, Germany
^b Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany

	ABSTRACT

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Despite intensive interest in understanding the differentiation of vascular smooth muscle cells (VSMC), no information is available about differential regulation of ion channels in these cells. Since expression of the L-type Ca²⁺ channel can be influenced by differentiation in other cell types, we tested the hypothesis that the L-type (C class) channel is a specific differentiation marker of VSMC and that expression of these channels depends on the state of cell differentiation. We used rat aortic (A7r5) VSMC, which express functional L-type Ca²⁺ channels, and induced dedifferentiation by cell culture in different media. Treatment with retinoic acid was used to redifferentiate the VSMC. We characterized the differentiated state of the cells by using immunohistochemistry and Western blot analysis for smooth muscle (SM) -actin and SM-myosin heavy chain (MHC). The number of functional Ca²⁺ channels was significantly decreased in dedifferentiated VSMC and increased upon differentiation with retinoic acid. Ca²⁺ channel function was assessed by whole-cell voltage clamp techniques. Using Western blot and dihydropyridine binding analysis, we found that the expression of the Ca²⁺ channel ₁ subunit, and to a lesser extent the ß₂ subunit, was directly correlated with the expression of SM -actin and SM-MHC. We conclude that expression of L-type Ca²⁺ channel ₁ subunits, and thus a functional Ca²⁺ channel, is highly coordinated with expression of the SM-specific proteins required for specialized smooth muscle cell functions. Furthermore, our results demonstrate that the L-type Ca²⁺ channel is a novel marker for differentiation of VSMC. The data suggest that regulation of ion channel expression during differentiation may have physiological importance for normal smooth muscle function and may influence VSMC behavior under pathophysiological conditions.—Gollasch, M., Haase, H., Ried, C., Lindschau, C., Morano, I., Luft, F. C., Haller, H. L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells. FASEB J. 12, 593–601 (1998)

Key Words: voltage-dependent Ca²⁺ channels • dihydropyridines • arterial smooth muscle • differentiation • retinoic acid • A7r5 cells • atherosclerosis

	INTRODUCTION

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VASCULAR SMOOTH MUSCLE cells (VSMC)² normally exist in a quiescent, differentiated state in the blood vessel wall. VSMC express a unique repertoire of contractile proteins, ion channels, receptors, and signaling molecules that are acquired during differentiation and are necessary for their contractile function. A major feature of chronic vascular diseases and atherosclerotic plaque development is the dedifferentiation of intimal VSMC with phenotypic changes and partial loss of their differentiated properties (1, 2). VSMC dedifferentiation seems to be a prerequisite for their subsequent migration and proliferation in the intima (3). The dedifferentiation shows a high degree of plasticity and is reversible in vitro (1). Although this phenomenon and its relevance for the pathogenesis of chronic vascular disease have been known for some time, only a few markers of VSMC differentiation have been described thus far. Dedifferentiation of human and experimental VSMC is associated with a decreased expression of smooth muscle (SM) contractile and cytoskeletal proteins including SM -actin, SM-myosin heavy chain (MHC), calponin, SM-22 , h-caldesmon, vinculin, and 20-kDa myosin light chains, as well as an increased expression of the nonmuscle variants of these proteins (2). The functional properties of the dedifferentiated cells are also altered; for instance, specific receptors such as angiotensin II are lost. We recently described a decrease in protein kinase C expression, indicating altered signal transduction in dedifferentiated VSMC (4).

Differentiated VSMC express a large repertoire of ion channels, including voltage-dependent dihydropyridine-sensitive L-type Ca²⁺ channels (5, 6). These channels are critical in regulating the cell's contractile behavior through effects on its electrical activities and sensitivity to stimulation by hormones and contractile agonists. Most ion channels are not expressed in the multipotential cells that give rise to VSMC, but rather appear during differentiation/maturation. Moreover, they may be `lost' during VSMC dedifferentiation. As such, ion channels may represent logical candidates for markers of VSMC differentiation. Ca²⁺ channels are particularly important in VSMC, where they are largely responsible for regulating VSMC contractility (4, 6). Regulation of voltage-dependent Ca²⁺ channels or other ion channels in VSMC differentiation has not been examined. We investigated the hypothesis that Ca²⁺ channel expression is a differentiation marker for VSMC and that channel expression is down-regulated during dedifferentiation. Since the Ca²⁺ channel is a multisubunit protein minimally consisting of ₁-, ₂/-, and ß subunits (7, 8), we measured the expression of L-type Ca²⁺ channels (by using Western blot analysis of the ₁-, ß₂-, and ß₃ subunits) and performed dihydropyridine (DHP) binding analysis in VSMC.

	MATERIALS AND METHODS

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Materials
Bay K 8644 and nimodipine were obtained from RBI (Natick, Mass.). EGTA (ethylene bis(oxyethylenenitrilo)tetraacetic acid), Hepes, and retinoic acid (RA) were purchased from Sigma-Aldrich (Deisenhofen, Germany). All salts were obtained from Merck (Darmstadt, Germany).

Cell culture
Rat aortic A7r5 cells (passage 10–30) were purchased from the American Type Culture Collection (Rockville, Md.). The cells were grown in Dulbecco's modified Eagle medium (DMEM) or M199 medium in the presence of 10% fetal calf serum (Gibco, Eggenstein, Germany), 1.7 mM L-glutamine, streptomycin (30 µg/ml), penicillin (30 units/ml), and nonessential amino acids at 37°C in humidified air with 5% CO₂ (9). The cells were cultured for more than 4 wk before use in the experiments.

Production and purification of antibodies
Rabbit polyclonal antibodies were generated against peptides corresponding to specific sequences of the _1C, ß₂, and ß₃ subunits in New Zealand white rabbits, as described previously (10). The peptides were commercially synthesized and purified (BioTeZ, Berlin-Buch, Germany). The purity was greater than 98% as assessed by high-performance liquid chromatography and mass spectroscopy. Antigenic epitopes comprised the amino acid sequence 799–817 (EEEEKERKKLARTASPEKK) of the cytoplasmic linker between repeats II and III of the _1C (10). This sequence is considered to be highly specific for the class C ₁ subunit and does not differ in the cardiac (10) and smooth muscle (11) protein. The antibodies specific for ß subunit isoforms of the Ca²⁺ channel were directed against the carboxyl-terminal 10 amino acid residues (EWNRDVYIRQ) of the ß₂ subunit (12) and the carboxyl-terminal 11 amino acid residues (QRNRPWPKDSY) of the ß₃ subunit (13). The resulting antibodies were affinity purified on peptide affinity columns (14). The anti-ß subunit antibodies have been characterized elsewhere (15, 16).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis
Frozen samples of Triton-solubilized A7r5 cells were thawed in 200 µl of SDS sample buffer containing 5% SDS, 50 mM Tris/HCl, pH 7.5, 250 mM sucrose, 75 mM urea, and 60 mM ß-mercaptoethanol, denaturated for 2 min at 95°C, and cleared by centrifugation at 10,000 g (Sorvall MC12V; DuPont) for 5 min. Proteins were separated by SDS-PAGE using 6.5% polyacrylamide gels at 4°C for 3 h with 30 mA constant current and electrophoretically transferred from SDS gels to nitrocellulose (Hybond-C, 0.45 µm, Amersham, Arlington Heights, Ill.) in a buffer containing 40 mM Tris, 300 mM glycine, 0.01% SDS, and 20% (v/v) methanol (2 h, 1.6 mA/cm²), using the Bio-Rad Mini-Protean II system. Nitrocellulose transfers were stained for protein with Ponceau S (Sigma) and further processed for Western blot analysis as described (15, 16). Briefly, the nitrocellulose transfers were incubated with the antibodies against either _1C, ß₂, ß₃, or -actin at a concentration of 0.5 to 1 µg IgG/ml for 90 min and the secondary peroxidase-conjugated antibody [anti-rabbit IgG, (BioGenes, Berlin, Germany), diluted 1:10.000] for 1 h at room temperature. Immunoreactive protein bands were visualized by an enhanced chemiluminescence reaction kit (ECL, Amersham, Braunschweig, Germany), using an X-ray film (X-Omat, Kodak, Rochester, N.Y.). The signals were scanned densitometrically with an Epson GT 8000 (Scan-Pack, Biometra, Göttingen, Germany). The blots were exposed for varying times to ensure that signals were in the linear range for densitometric analysis. To normalize for possible differences in the amount of protein loaded onto gels or blotted to nitrocellulose, the ₁ and ß subunit band intensities were normalized to that of a prominent 45-kDa Ponceau-stained band.

1,4-Dihydropyridine binding
For binding experiments, cells were washed twice with 50 mM Tris/HCl-buffer, pH 7.4 (buffer A), harvested by gentle scraping, sedimented, and frozen at -80°C. Cell pellets were thawed in buffer A containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM pepstatin A. Equilibrium binding of isradipine {(+)-[³H]methyl-PN200–110; Amersham, spec. act. of 83 Ci/mmol} was performed in triplicate at 37°C for 30 min, as described (17), with the modification that the cells were depolarized. The binding reaction was started by adding the suspended cells (200 µg protein) to 2 ml medium, resulting in a final concentration of 50 mM Tris/HCl-buffer, pH 7.4, 100 mM KCl, 1 mM CaCl₂, 0.1 mM PMSF, and 0.1 nM (+)[³H]PN200–110. Nonspecific binding was determined in the presence of 1 µM nitrendipine. DHP binding assays were performed using at least three different populations of A7r5 cells.

Patch clamp experiments
Ca²⁺ channel currents were recorded as previously described (18, 19) in the whole-cell configuration (20) by using a List patch-clamp amplifier EPC 7. Data acquisition and command potentials were controlled with commercial software programs using a CED1401 interface (Cambridge Electronic Design Ltd., Cambridge, U.K.). Currents were recorded from holding potentials of -80 mV (-100 mV) during linear voltage ramps at 0.67 V/s from -100 mV to +80 mV or 300 ms step pulses to different potentials; pulse frequency, 0.33 Hz. Analysis of whole-cell currents was performed using CED Patch and Voltage Clamp Software Version 6.08 (Cambridge Electronic Design Ltd., Cambridge, U.K.). Ba²⁺ was used as charge carrier; K⁺ currents were blocked by Cs⁺. In most experiments, the bath solution contained (in mM) NaCl 125, BaCl₂ 10.8, MgCl₂ 1, CsCl 5.4, glucose 10, and Na-Hepes 10 (pH 7.4 at 37°C). The patch pipette was filled with a solution containing (in mM) CsCl 120, MgCl₂ 3, Mg-ATP 5, EGTA 10, and Cs-Hepes 5 (pH 7.4 at 37° C). The resistance of the pipettes was 4 to 6 MOhm. Experiments were performed at 22–24°C. For additional technical details, see Gollasch et al. (18, 19).

The voltage-dependence of the steady-state inactivation of the Ca²⁺ channel currents could be described by a Boltzmann equation of the form:

where I is the amplitude of inward Ca²⁺ channel current elicited by test pulse after conditioning prepulse (V) with a duration of 1.5 s between -80 mV and +40 mV, I_max is the amplitude of inward Ca²⁺ current elicited by test pulse from a conditioning prepulse of -80 mV, C is the noninactivating current, V_h is the value of 1.5 s conditioning potential causing 50% inactivation of the Ca²⁺ channel current, and k is the steepness factor characterizing the voltage sensitivity of the channels.

Immunocytochemistry
The techniques for confocal microscopy were as described previously (4, 21). Cells were fixed with 4% paraformaldehyde and permeabilized with 80% methanol at -20°C. After incubation with 3% skimmed milk in a phosphate-buffered solution (PBS) for 60 min, the preparation was incubated for 1 h at room temperature with the myosin antibody [monoclonal anti-cytoskeletal myosin (1:20), clone 2F12. A9; Dianova, Hamburg, Germany] or a monoclonal anti-smooth muscle cell -actin (1:400, clone 1A4; Makor Renner, Darmstadt, Germany). The antibodies were diluted in PBS with 0.1% bovine serum albumin (BSA) (1:80); the cells were washed three times with PBS and then exposed to the secondary antibody (Cy2-or Cy3-conjugated anti-rabbit or anti-mouse IgG, at 1:100, 1% BSA/PBS (Dianova, Hamburg, Germany) for 60 min. The preparation was mounted with 50% glycerol under a glass coverslip on a Nikon-Diaphot (Tokyo, Japan) microscope. A Biorad MRC 600 confocal imaging system (Bio-Rad Laboratories, Freiburg, Germany) with an argon/krypton laser was used. At least 30 cells from each of at least three experiments were examined under each experimental condition. The results were reproduced by two separate investigators.

Statistical analysis
All values are given as mean ± SEM. The term n represents the number of cells tested. The Wilcoxon rank sum, analysis of variance, or Mann-Whitney-Wilcoxon test was used to determine significant differences. A value of P < 0.05 was considered statistically significant.

	RESULTS

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We first characterized the expression of known marker proteins for differentiation in the cultured A7r5 cells. These results are shown in Fig. 1. When VSMC were cultured in DMEM, the cells displayed a differentiated phenotype with abundant expression of -actin stress fibers (panel A, upper lane) and smooth muscle-specific MHC (panel A, lower lane). These protein markers of differentiation were markedly reduced when the cells were changed to medium M 199. After 7 days in M 199, -actin stress fibers and SM-MHC were barely detectable. No differences in cell number or cell shape under the light microscope were observed (data not shown). The loss of these differentiation markers was reversible when cell culture conditions were changed to DMEM (data not shown). In previous experiments we demonstrated that RA is a strong inductor of VSMC differentiation. When dedifferentiated A7r5 cells were exposed to RA, they showed a strong increase in the expression of -actin and SM-MHC expression, indicating RA-induced differentiation ( Fig. 1A). The dedifferentiation of VSMC cells by different culture conditions—namely, the loss of the differentiation markers -actin and SM-MHC and the subsequent differentiation induced by RA—was confirmed by Western blotting ( Fig. 1B). Densitometric analysis of the Western blots demonstrated a significant loss of -actin in M 199 compared to DMEM and an increase after RA.

View larger version (118K): [in this window] [in a new window]   Figure 1. A) Immunohistochemistry for -actin and SM-MHC of VSMC during different culture conditions (DMEM and M199) and after treatment with retinoic acid (RA 10-8 M) for 48 h. B) Western blot for VSMC -actin during different culture conditions and after treatment with RA for 48 h. VSMC in DMEM showed a strong expression of actin stress fibers and myosin. Exposure to M 199 led to dedifferentiation, with decreased expression of both -actin and myosin. Treatment of dedifferentiated VSMC with RA induced a strong increase in actin and myosin expression. *P < 0.05.

Using the patch clamp method, we then analyzed the Ca²⁺ channel currents in the VSMC under different culture conditions and after treatment with RA. The whole-cell inward current was characterized using 10 mM Ba²⁺ as charge carrier; the holding potential was -80 mV. A7r5 cells cultured in DMEM, M199, or M199 plus RA exhibited slowly inactivating Ca²⁺ channel currents when pulsed to a test potential of 0 mV. Fast inactivating (T type) currents were observed only in 23% of all cells tested (n=65). To estimate the current–voltage relations (IV curves), the test potential was linearly varied from -100 mV to 80 mV (or 100 mV). Figure 2A shows recordings from typical control cells cultured in DMEM, M199, or M199 plus RA, demonstrating the Ca²⁺ channel current characteristics. The recorded currents were U-shaped in all cells. However, the peak Ca²⁺ channel current was augmented threefold in cells cultured in DMEM vs. cells cultured in M199. Figure 2B illustrates that mean maximal currents were 614 ± 86 pA (n=29) and 211 ± 37 pA (n=23) in cells cultured in DMEM and M199, respectively. Redifferentiation of A7r5 cells cultured in M199 by RA (10^-8 M) increased the peak Ca²⁺ channel current to 467 ± 75 pA (n=13). Peak Ca²⁺ channel currents in DMEM, M199, and M199 plus RA were significantly different (P<0.01) ( Fig. 2A, B). The cell membrane capacitances were not different in the three A7r5 cell groups, indicating that the changes in peak Ca²⁺ channel currents observed were due to differences in channel density but not in cell size. The cell membrane capacitances were 78 ± 8 pF (n=22), 76 ± 11 pF (n=22), and 70 ± 5 pF (n=13) in cells cultured in DMEM, M199, and M199 plus RA (10^-8 M), respectively.

View larger version (32K): [in this window] [in a new window]   Figure 2. Functional, active L-type Ca2+ channel currents in single vascular smooth muscle A7r5 cells during different culture conditions (DMEM and M199) and after treatment with RA (10-8M) for 48 h. A) Recordings of Ca2+ channel currents (ICa) of typical cells. Holding potential, -80 mV; linear voltage ramps at 0.67 V/s from -100 mV to +80 mV; pulse frequency, 0.33 Hz. In all cells, ICa is augmented by 1 µM Bay K 8644 and can then be washed out. Subsequent application of 1 µM nimodipine blocked ICa. Con, control; Bay K, Bay K 8644 (1 µM); Nimo, nimodipine (1 µM). B) Peak ICa recorded in A7r5 cells cultured in DMEM (n=29), M199 (n=23), or M199 plus RA (n=13). C) Effect of 1 µM Bay K 8644 on peak ICa in A7r5 cells cultured in DMEM (n=7), M199 (n=5), or M199 plus retinoic acid (n=5). Percentage changes in peak ICa are shown.

No differences were detected among the three cell groups in apparent threshold potentials, potentials at which maximal currents occurred, or reversal potentials. The apparent thresholds occurred at -32 ± 3 mV (n=9), -27 ± 2 mV (n=16), and -34 ± 3 mV (n=11) in cells cultured in DMEM, M199, and M199 plus RA (10^-8 M), respectively. The potentials with peak Ca²⁺ currents were 5 ± 2 mV (n=19), 9 ± 2 mV (n=20), and 3 ± 2 mV (n=13) in cells cultured in DMEM, M199, and M199 plus RA, respectively. The reversal potentials were 56 ± 2 mV (n=19), 53 ± 3 mV (n=20), and 55 ± 3 mV (n=13) in cells cultured in DMEM, M199, and M199 plus RA, respectively. The latter may be underestimated because of a possible Cs⁺ flux through Ca²⁺ channels at positive potentials.

The Ca²⁺ channel currents were reversibly blocked by the DHP antagonist nimodipine (1 µM) or Cd²⁺ (100 µM) ( Fig. 2A). They were reversibly increased by the DHP agonist Bay K 8644 (1 µM). These results indicate functional activity of L-type channels in A7r5 cells. To maximally stimulate Ca²⁺ channels, we used Bay K 8644 at a submaximal dose of 1 µM. As shown in Fig. 2B, 1 µM Bay K 8644 induced an identical percent increase in peak Ca²⁺ channel current in cells cultured in DMEM, M199, or M199 plus RA (10^-8 M). This finding supports the suggestion that the density of functional L-type channels is higher in differentiated A7r5 cells cultured in DMEM or M199 plus RA (10^-8 M) than in dedifferentiated A7r5 cells in M199.

We then tested whether or not the increase in Ca²⁺ currents in differentiated VSMC was accompanied by an increase in Ca²⁺ channel expression. In an initial set of experiments, we estimated the 1,4-dihydropyridine binding capacity in depolarized VSCM cells by using 100 pM of [³H](+)PN200–110, a concentration that is expected to saturate the high-affinity DHP binding site in A7r5 cells. Under these conditions, DMEM-cultivated A7r5 cells displayed a significantly (P<0.05) higher density of receptor sites compared to M199-treated cells (21±3 fmol/mg protein [n=4] vs. 9 ± 2 fmol/mg protein [n=4]). Addition of RA to M199-cultivated cells increased the DHP binding to 17 ± 3 fmol/mg protein (n=3), not significantly different from VSCM cultivated in DMEM. Thus, binding data suggested that the high-affinity DHP receptor expression is up-regulated in differentiated A7r5 cells. The Ca²⁺ channel is a multisubunit protein consisting minimally of ₁, ₂/, and ß subunits. Because it is not yet known whether the increase in DHP binding, as well as in Ca²⁺ currents, reflects an up-regulation of the channel-forming ₁ protein or changes in the auxiliary subunits, we analyzed the expression of the putative Ca channel subunits in VSCM, _1C, ß₂, and ß₃ in more detail using affinity-purified, isoform-specific antibodies. The anti-_1C antibody directed against an internal sequence of class C ₁ subunits reacted strongly with a single 250-kDa protein on Western blots of A7r5 cells ( Fig. 3A). Immunostaining was completely prevented after preincubation of the antibody with the antigenic peptide ( Fig. 3A).

View larger version (33K): [in this window] [in a new window]   Figure 3. Expression of the Ca2+ channel subunits 1C and ß2 in A7r5 cells during different culture conditions. A) A7r5 cells after cultivation in DMEM, M199, and M199 plus retinioc acid were probed with the anti-1C and anti-ß2 antibody. Immunostaining for 25 µg (left lane) and 50 µg protein (right lane) is shown. Nonspecific staining was determined after preincubation of the antibodies with 1 µM of the antigenic peptide, as indicated (+peptide). B) Bar graphs depicting relative values for 1C and ß2 immunostaining. Densitometric values for immunostaining were related to the staining intensity obtained with DMEM-cultivated cells. Values are means ± SEM from six (1C) and four (ß2) independent experiments. *P < 0.01.

In A7r5 cells, immunoblot analysis using the affinity-purified anti-ß₂ antibody identified a doublet in the M_r range of 75–80 kDa ( Fig. 3B). This apparent molecular mass for the ß₂ produced in A7r5 cells is consistent with the M_r of the cardiac ß₂ subunit enriched from the animal model (16). The sharp band migrating with an M_r of ~100 kDa appeared to be nonspecific, since the immunorecognition was not efficiently inhibited by the antigenic peptide ( Fig. 3A). The anti-ß₃ antibody stained weakly an ~55-kDa protein on Western blots of A7r5 cells, which is consistent with the M_r expected for the ß₃ isoform (data not shown).

To examine whether or not the expression level of Ca²⁺ channel subunits changed during differentiation of A7r5 cells, identical amounts of solubilized protein from the experimental groups were subjected to Western blot analysis and probed with subunit-specific antibodies. The representative experiment shown in Fig. 3A illustrates differences in the amount of immunoreactive proteins for both _1C and ß₂ in A7r5 cells cultivated under different conditions, whereas the ß₃ staining did not differ between the experimental groups (data not shown). Pooled data from several experiments revealed that the _1C was significantly reduced by about 50% in M199-cultivated cells compared to those with DMEM, whereas M199 complemented with RA caused a slight, but not significant, reduction in _1C ( Fig. 3B). Similar results were obtained for the expression level of the ß₂ subunit: the ß₂ immunoreactivity in A7r5 cells cultivated with DMEM was about twofold higher than in M199-cultivated cells irrespective of the presence of RA ( Fig. 3B).

To determine whether differences in Ca²⁺ channel ₁ subunit and ß₂ subunit expression were accompanied by differences in functional Ca²⁺ channels, we further analyzed steady-state inactivation curves of whole-cell Ca²⁺ channel currents by using the patch clamp method. Steady-state inactivation curves were determined by using a 1.5 s prepulse recording protocol that allowed estimation of the potential (V_h), causing 50% inactivation of the channels, and estimation of the steepness factor (k) characterizing the voltage sensitivity of the channels ( Fig. 4). Although k values were not different among the three groups of cells, V_h was significantly smaller in cells cultured in DMEM rather than M199 ( Fig. 4). Addition of RA (10^-8 M) to the dedifferentiated cells in M199 did not restore the V_h value of cells, indicating incomplete restoration of channel inactivation properties during redifferentiation induced by RA.

View larger version (20K): [in this window] [in a new window]   Figure 4. Steady-state inactivation of ICa in A7r5 cells cultured in DMEM (n=8), M199 (n=9), or M199 plus retinoic acid (n=10). Peak current (I/Imax) is plotted against conditioning prepotential (V). Currents were recorded during a test pulse to 0 mV, which was preceded by conditioning membrane potentials varying from -70 mV to +10 mV of 1.5 s. Smooth curves are the best fits according to the Boltzmann equation described in Materials and Methods. I/Imax characterizes the current availability of ICa. Half-maximal inactivation (Vh) occurred at -31.3 mV, -21.5 mV, and -23.2 mV in cells cultured in DMEM, M199, and M199 plus retinoic acid (RA), respectively. The steepness factors (k) were 8.1 mV, 8.1 mV, and 8.5 mV in cells cultured in DMEM, M199, and M199 plus rRA, respectively.

	DISCUSSION

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We report here the first analysis of the expression of the voltage-dependent L-type Ca²⁺ channel in different phenotypic states of VSMC. L-type Ca²⁺ channels play an important role in vascular smooth muscle cells for physiological functions and as therapeutic targets. The novel finding of this study is that the expression of functional DHP-sensitive (C class) L-type Ca²⁺ channels, estimated by Western blot analysis and characterized by whole-cell voltage clamp experiments, correlates with the phenotypic state of VSMC differentiation. Ca²⁺ channel currents are larger in differentiated VSMC than in dedifferentiated cells, and conversion of the cell phenotype from the dedifferentiated into the differentiated state by RA treatment led to an increase in the (C class) L-type Ca²⁺ channel current. We also observed that submaximal concentrations of Bay K 8644 induced an identical percent increase in peak Ca²⁺ channel current in all cells, independent of the phenotypic state of cell differentiation. The cell membrane capacitances, as indicators of cell sizes, were not different among the phenotypically different cells. Together, these results suggest that the density of functional L-type, Ca²⁺ channels is high in differentiated but low in dedifferentiated VSMC.

Induction of the L-type Ca²⁺ channel during differentiation has also been observed in other cell types. Neuronal cells have mostly been investigated so far. Reuter and co-workers (22, 23) have shown that differentiation of PC12 cells is associated with an increase in voltage-dependent Ca²⁺ currents. Similar observations have been made by others (24–27). An association between Ca²⁺ channel expression and differentiation has been observed in other cell types. In the rat embryo, the number of L-type Ca²⁺ channel binding sites is low at the fetal stage and increases drastically during postnatal development of skeletal and cardiac muscle (28). In a cardiac cell line, Caviedes et al. (29) demonstrated increased voltage-dependent Ca²⁺ currents during differentiation.

We observed that the increase in Ca²⁺ channel currents was accompanied by an increase in DHP receptor protein as assessed by ligand binding studies, making an increase in the number of L-type channel ₁ subunits likely. The ₁ subunit has been shown to be the pore-forming subunit, drug receptor, and voltage sensor, and is the `signature' subunit that determines many basic characteristics of the different types of the channel. Molecular cloning has identified the C class ₁ subunits (CaCh2), which encode the L-type DHP-sensitive and omega-conotoxin-insensitive Ca²⁺ channels (7, 30). In accordance with these data, our Western blot analysis shows that expression of (C_b) Ca²⁺ channel ₁ subunits is high in differentiated but low in dedifferentiated VSMC. Expression of this subunit has not been investigated extensively (31). Varadi et al. (32) reported that the expression of C class ₁ and ₂ mRNAs is developmentally regulated in differentiating C2C12 myogenic cells. The (C class) ₁ subunit mRNA is not detectable in the myoblast form of C2C12 cells whereas its expression is induced by 20-fold in differentiated myotubes. Induction of the ₁ subunit by ß-adrenergic agonists was demonstrated in cardiomyocytes by Maki et al. (33). Biel et al. (8) demonstrated that (C class) ₁, ß, and mRNA are expressed together in differentiated but not in proliferating BC3H1 cells. They suggested that different splice variants of the genes for the ₁, ß, and subunits exist in tissues containing L-type Ca²⁺ channels and that their expression is regulated in a coordinate manner. However, our results in vascular smooth muscle cells do not support the latter assumption. In our experiments, expression of ß₂ and ß₃ subunits were not highly coordinated with the ₁ subunits during differentiation.

Our results shed new light on the interaction of the different channel subunits in vascular smooth muscle cells. DHP targeting to the ₁ subunit as well as formation and stabilization of the complex (C class) L-type Ca²⁺ channel are significantly influenced by accessory Ca²⁺ channel ß subunits (34–37). For example, the ß₂ subunit has been shown to increase the amount of C class ₁ subunits at the plasma membrane (35). This ß subunit and the ß₃ subunit are widely distributed, even in smooth muscles (8, 38). Using Western blot analysis, we found that expression of the Ca²⁺ channel ß₃ subunit was similar in different phenotypic states of VSMC. In contrast, expression of the ß₂ subunit was high in differentiated VSMC cultured in DMEM, but low in dedifferentiated VSMC cultured in M199. Redifferentiation of VSMC cultured in M199 by RA treatment led to an increase in SM -actin and SM-MHC to levels observed in VSMC cultured in DMEM, but did not induce an elevation of the expression of ß₂ subunits. Several reports have demonstrated that these subunits can play multiple roles in the formation, stabilization, gating, drug targeting, and modulation of the channel complex (34–37). Because ß subunits induce a shift of inactivation curves to more negative membrane potentials (8, 34), the data on ß₂ subunit expression are also in accordance with our analysis of Ca²⁺ channel inactivation. The analysis shows that inactivation curves are the same in cells cultured in M199 or M199 plus RA, with similar low levels of ß₂ subunits. In contrast, the data show that half-maximal inactivation was shifted to more negative membrane potentials (V_h, -31.3 mV) in A7r5 cells in DMEM with high levels of ß₂ subunits. We therefore conclude that expression of the Ca²⁺ channel ₁ subunit, but not of ß₂ and ß₃ subunits, is highly coordinated with expression of the SM-specific proteins necessary for specialized smooth muscle functions.

The present study raises new questions about the role of intracellular Ca²⁺ ions in the process of differentiation. One could speculate that expression of the L-type Ca²⁺ channel itself plays a major role in the differentiation process. We cannot answer this question yet; however, the intracellular Ca²⁺ concentration and Ca²⁺ influx are important in cell differentiation. Gillo et al. (39) showed that Ca²⁺ influx is an early event associated with differentiation in murine erythroleukemia cells; blockade of this Ca²⁺ influx inhibits induced differentiation. Luo and co-workers (40) observed that intracellular, ryanodine-sensitive Ca²⁺ channels and extracellular, L-type Ca²⁺ channels play an important role during differentiation from myoblasts to myotubes. Support for a critical role of Ca²⁺ influx in cell differentiation also comes from the work of MacVicar (41) and others (42, 43), who showed that blockade of Ca²⁺ influx inhibited differentiation in neuronal cells completely. The increased Ca²⁺ influx during differentiation could by itself lead to an increased expression of L-type Ca²⁺ channels. In skeletal muscle, Navarro (44) observed that expression of dihydropyridine receptors is modulated by the intracellular Ca²⁺ concentration.

Our results suggest that expression of the L-type Ca²⁺ channel is a late step in the differentiation of vascular smooth muscle cells. Differentiation of vascular smooth muscle cells is associated with increased expression of SM -actin, SM-MHC, calponin, SM-22 , h-caldesmon, vinculin, 20-kDa myosin light chains, and the nonmuscle variants of these proteins (for a review, see ref 2). Most are markers of the early stages of differentiation. The L-type, voltage-dependent Ca²⁺ channel is the first example of a late marker and may therefore be a useful marker for investigating VSMC under pathophysiological conditions (4). However, extrapolations from our results to the the in vivo situation of pathologically altered VSMC must be drawn with caution. A7r5 cells are a permanent cell line derived from rat thoracic aorta and are prone to obtain different patterns of gene expression and protein regulation compared to `wild-type' VSMC. However, A7r5 cells have been used to assess VSMC function for more than 13 years (45–47). This cell line was especially important in describing the intracellular Ca²⁺ regulation in vascular smooth muscle cells and Ca²⁺ channels; Ca²⁺ transport systems and intracellular Ca²⁺ stores have been investigated extensively in these cells (45–47). Several investigators have compared the mechanisms in A7r5 cells to freshly isolated VSMC and obtained similar results (45, 47). We believe, therefore, that the database on A7r5 cells justifies their use as a model of VSMC and a tool to investigate specific mechanisms of differentiation.

In conclusion, our results demonstrate that expression of the L-type, Ca²⁺ channel ₁ subunit is highly coordinated with expression of the SM-specific proteins necessary for specialized smooth muscle functions. Furthermore, expression of this subunit determines the number of functional L-type channels in different phenotypic states of VSMC. Moreover, our results suggest that the (C_b) L-type Ca²⁺ channel ₁ subunit is a novel marker for differentiation of VSMC. Regulation of ion channel expression during differentiation may have physiological importance for normal smooth muscle function and may influence smooth muscle behavior in pathophysiological conditions such as the formation and evolution of atherosclerotic plaques in arteries.

	ACKNOWLEDGMENTS

 
We thank Dr. R. Bychkov for critically reading the manuscript and Petra Quass and Jana Czychi for expert technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.

	FOOTNOTES

 
¹ Correspondence: Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, Germany. E-mail: haller{at}mdc-berlin.de

² Abbreviations: MHC, myosin heavy chain; DHP, dihydropyridine; RA, retinoic acid; DMEM, Dulbecco's modified Eagle medium; SDS-PAGE, sulfate-polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered solution; BSA, bovine serum albumin; EGTA, ethylene bis(oxyethylenenitrilo)tetraacetic acid; VSMC, vascular smooth muscle cells; SM, smooth muscle;

Received for publication January 31, 1997. Accepted for publication January 2, 1998.

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