Cell Cycle-Dependent Expression of L- and T-Type Ca2+ Currents in Rat Aortic Smooth Muscle Cells in Primary Culture
The expression of L- and T-type Ca2+ channels has been reported to change during various biological events, including cellular differentiation and proliferation. The present study aimed to examine whether or not the expression of L- and T-type Ca2+ channels depends on the cell cycle in rat aortic smooth muscle cells in primary culture. Both the phase of the cell cycle and the functional expression of Ca2+ channels were determined in the same single cell, using an immunocytochemical analysis of cell cycle-specific nuclear antigens and a whole-cell voltage-clamp method, respectively. In the G0 (n=130) and M (n=75) phases, all cells showed only L-type Ca2+ currents. The cells showing a T-type Ca2+ current appeared in the G1 phase (37%, n=85) and increased in the S phase (90%, n=21). For L-type Ca2+ channels, the current density was significantly greater in the G1 phase than in the G0 and M phases. However, either the voltage-dependent properties or the dose-response relationships of Bay K 8644- and second messenger-induced modulations of L-type Ca2+ current did not differ in the four phases of the cell cycle. These findings thus indicate that the expression of L- and T-type Ca2+ channels depends on the cell cycle, whereas the characteristics of L-type Ca2+ channels do not differ between the phases of the cell cycle.
Two types (L- and T-type) of Ca2+ channels have been previously identified in various preparations. Expression of the types of Ca2+ channels changes with cellular differentiation, proliferation, or hypertrophy.1 2 3 4 5 6 7 8 9 10 11 In neurons,1 2 3 cardiac myocytes,4 5 and skeletal muscle cells,6 7 T-type Ca2+ channels are predominantly expressed at the early embryonic or neonatal stage; thereafter, they disappear with maturation. Moreover, T-type Ca2+ channels are reexpressed in adult myocytes when they are stimulated to an active growth phase by pressure overload8 or growth hormone.9 Rat aortic smooth muscle cells in primary culture show heterogeneous expression of L- and T-type Ca2+ channels: some cells express only one type of Ca2+ channel, whereas others express both L- and T-type Ca2+ channels.10 11 12 13 14 Some of those studies have also suggested that cells in the proliferative phases frequently express T-type Ca2+ channels.10 11 In contrast, most cells in the nonproliferative phases express only L-type Ca2+ channels. These findings support the hypothesis that expression of L- and T-type Ca2+ channels may differ between the phases of the cell cycle. Recent studies have suggested that expression of Cl− channels15 16 and K+ channels17 18 differs depending on the cell-cycle phase, but the cell cycle-dependent difference in expression of Ca2+ channels has not been previously demonstrated.
The present study thus examined whether expression of L- and T-type Ca2+ channels differs between the phases of the cell cycle of rat aortic smooth muscle cells in primary culture. We recently developed a method to determine the phase of the cell cycle in an individual vascular smooth muscle cell.19 In the present study, we determined the phase of the cell cycle and the type of Ca2+ channels expressed in the same single cell. We also examined whether the characteristics of L-type Ca2+ channels differed between the phases of the cell cycle.
Materials and Methods
Smooth muscle cells of the aortic media in male Wistar rats (250 to 300 g) were dispersed enzymatically and cultured on a chamber slide (Lab-Tek), as previously described.19 It was confirmed by electron microscopic examination and direct immunofluorescence stainings of smooth muscle myosin and actin that the cells were not contaminated by fibroblasts or endothelial cells.
The experimental conditions under which current recordings were made were similar to those reported in our previous studies.10 12 13 14 Briefly, the cultured cells on a plate (No. 4804, Lab-Tek) were trypsinized for 1 minute and then were placed in a recording chamber. We performed the whole-cell voltage-clamp method using heat-polished glass patch electrodes (VC-H075P, Termo) with a tip resistance of 2 to 5 MΩ to evaluate the functional expression of L- and T-type Ca2+ channels. The currents were amplified using a patch-clamp amplifier (EPC-7, List-Electronic) with capacitance and series-resistance compensation and filtered at 2.0 kHz. All experiments were monitored on a digital storage scope (DS-6121A, Iwatsu) and stored on a PCM data recorder (RP-880, Sony) for later analysis. The amplitudes of ICa were measured at the peak of each current. The amplitudes of ICa were estimated by subtracting leakage currents from recording currents. Leakage current was calculated with the current elicited by a small hyperpolarizing pulse.
We estimated the peak amplitude of the T-type ICa by step depolarization to −30 mV from a VH of −100 mV. This step depolarization induced little or no activation of L-type Ca2+ channels.
When we estimated the peak amplitude of L-type ICa, we applied a step depolarization to 20 mV from a VH of −60 mV. This step depolarization induced little or no activation of T-type ICa. Preliminary studies using Cd2+ (0.1 mmol/L) or F− (5 mmol/L), which abolished L-type ICa but rarely affected T-type ICa,10 confirmed that the two protocols of step depolarizations were appropriate for separating the two types of ICa in rat aortic smooth muscle cells in primary culture. We could thus estimate the peak amplitude of both types of ICa separately by using the two-step depolarization protocols.10 12
The steady state inactivation of L-type Ca2+ channels was obtained using a double-pulse protocol: from a VH of −80 mV, the membrane was depolarized to different potential levels by a prepulse (3 seconds) before the constant test pulse (300 milliseconds) to 20 mV. The steady state activation of L-type Ca2+ channels was obtained by extrapolating the linear part of the corresponding I-V curve. The steady state inactivation and activation curves were deduced from the Boltzmann equation.
The internal solution contained (mmol/L) N-methyl-d-glucamine 110, Na2ATP 5, MgSO4 5, TEA-Cl 20, HEPES 5, EGTA 10, and Tris base 2. The pH was adjusted to 7.2 by HCl. The external solution contained (mmol/L) CaCl2 20, NaCl 110, KCl 2, glucose 10, TEA-Cl 15, 4-aminopyridine 5, and HEPES 5. The pH was adjusted to 7.4 with Tris base. During the experiments, the chamber (0.5 mL) was continuously perfused with external solution at a rate of 1 mL/min. When we examined the effects of drugs such as Bay K 8644, the external solution containing drugs was perfused at a rate of 10 mL/min. The exchange of the external solution was accomplished within 30 seconds. All experiments were carried out at room temperature (20°C to 24°C).
Immunocytochemical Analysis of the Cell Cycle
After recording ICa, we removed the electrode from the cell and put a mark on the slide (Lab-Tek) around the cell with a needle. Then the cell-cycle phase of the cell enclosed by the mark was determined as described previously.19 Briefly, the cell on the Lab-Tek slide was rapidly fixed in −20°C ethanol (75%) for 30 minutes and was subsequently washed with and incubated in phosphate-buffered solution containing 1 mg/mL bovine serum albumin for 10 minutes at 25°C. Double-labeled immunofluorescence staining was carried out by incubation with monoclonal antibodies against cell cycle-specific nuclear antigens (fluorescein isothiocyanate-conjugated anti-PCNA and R-phycoerythrin-conjugated Ki-67) for 30 minutes at 25°C. We observed the cell with double staining under a fluorescence microscope (Axioskop, Zeiss) and photographed it for later analysis. Because the staining properties of PCNA (no expression in the G0 phase, moderate expression in the G1 or G2 phase, and maximal expression in the S phase) and Ki-67 (no expression in the G0 phase, weak and aggregated expression in the G1 phase, increasing expression in the S phase, and maximal expression in the G2 and M phases) were different among the cell-cycle phases; the color pattern of the nucleus produced by double staining differed.20 The cells in the G0 phase had no specific nuclear fluorescence, whereas the cells in the G1 phase showed green fluorescence (PCNA) associated with aggregated spotty red-orange fluorescence (Ki-67). The cells in the M phase had red (Ki-67) fluorescence, and the cells in the S phase emitted both red (Ki-67) and green (PCNA) fluorescence, thus giving them a yellow appearance. Differentiated nuclear stainings were confirmed in vascular smooth muscle cells, which were arrested in the S or M phase by aphidicolin or TN-16, respectively.
In the present study, we determined the functional expression of Ca2+ channels using the whole-cell voltage-clamp method on 646 cells. In 338 of these 646 cells, we tried to determine the cell-cycle phase by an immunocytochemical analysis, and the cell-cycle phase could be determined in 311 of 338 cells. In the remaining 27 cells, the cell-cycle phase could not be determined because the cells were lost from the Lab-Tek slide during the fixation and staining procedures.
PDBu, dibutyryl cGMP, and dibutyryl cAMP were purchased from Sigma Chemical Co. Bay K 8644 and nifedipine were purchased from Wako Jun-yaku Kogyo. All drugs were prepared as a stock solution and were made to a final concentration with the external solution. Bay K 8644 and PDBu were dissolved in DMSO to make a stock solution. At the concentrations used (<0.1%), DMSO had no effect on L-type ICa. The monoclonal antibodies (fluorescein isothiocyanate-conjugated anti-PCNA and R-phycoerythrin-conjugated Ki-67) were purchased from Dako Japan.
The data are expressed as mean±SEM. The cell population concerning the expression of L- or T-type ICa was then compared among the different cell-cycle phases using the χ2 test. The current density of L- or T-type ICa of the cell was compared among the different cell-cycle phases by a one-way ANOVA with Bonferroni's test. V0.5 and slope factors in steady state inactivation or activation were compared by one-way ANOVA. The dose-response relationships of Bay K 8644-induced, PDBu-induced, or the dibutyryl cGMP-induced changes in L-type ICa were compared by two-way ANOVA. Values of P<.05 were considered to be significant.
Cell Cycle-Dependent Expression of L- and T-Type ICa
Rat aortic smooth muscle cells in primary culture express two distinct types (L- and T-type) of Ca2+ channel that can be determined by their characteristics, including voltage dependences, current kinetics, ion selectivities, sensitivities to various organic and inorganic Ca2+ antagonists, and modulation by various drugs.10 12 13 14 Under our experimental conditions, T- and L-type ICa can only be identified by differences in their voltage dependences and current kinetics, as previously described.10 In brief, L-type ICa can be identified by the findings that step depolarization to >−30 mV evokes a long-lasting type ICa from a VH of −60 mV and that ICa reaches the peak by step depolarization to ≈20 mV (Fig 1a, 1b, and 1d⇓⇓⇓). In contrast, T-type ICa can be identified by the findings that transient ICa is evoked by depolarization to >−60 mV from a VH of −100 mV and that ICa reaches the peak by depolarization to ≈−30 mV (Fig 1b and 1c⇓⇓). Three types of the cells were identified on the basis of the expression of the Ca2+ channels: cells expressing only L-type ICa (Fig 1a and 1d⇓⇓), those expressing both L- and T-type ICa (Fig 1b⇓), and those expressing only T-type ICa (Fig 1c⇓). The immunocytochemical analysis showed that the cell in panel a was in the G0 phase, that in panel b was in the G1 phase, that in panel c was in the S phase, and that in panel d was in the M phase.
Fig 2a⇓ summarizes the population of the cells showing L- and T-type ICa in each cell cycle. The total number of cells examined was 311. All cells in the G0 phase (n=130) showed only L-type ICa. In the G1 phase (n=85), most cells (63%) showed only L-type ICa; however, the remaining (37%) showed T-type ICa. In the S phase (n=21), most cells showed T-type ICa (90%). In the M phase (n=75), however, most cells (96%) showed only L-type ICa, whereas the few remaining cells (4%) had no detectable ICa. Thus, the expression of two types of Ca2+ channel depended on the phase of the cell cycle (P<.01). The current density of T-type ICa was significantly greater in the S phase than the G1 phase (P<.001) (Fig 2b⇓). The current density of L-type Ca2+ channels was significantly greater in the G1 phase than the G0 and M phases (P<.005), but there was no significant difference in the current density of L-type Ca2+ channels among the G0, M, and S phases (Fig 2c⇓).
Expression of L- and T-Type ICa and the Duration of Cell Culture
Previous studies have demonstrated that the expression of L- and T-type ICa changes with the duration of the cell culture.10 11 Fig 3⇓ shows the relationship between the duration of the culture and the population (percentage) of the cells showing T-type ICa of a total of 646 cells. Within 6 hours (the “zero” culture day in Fig 3⇓), all cells showed only L-type ICa (n=35). The immunocytochemical analysis of the cell cycle, which was performed in 25 of 35 cells, showed that all 25 cells were in the G0 phase. From 6 to 24 hours (the 6-hour culture day in Fig 3⇓), 20 cells showed only L-type ICa, and three cells showed both L- and T-type ICa. The immunocytochemical analysis of the cell cycle of those cells, which was performed in 21 of 23 cells, showed that the cells showing only L-type ICa were in the G0 phase (n=18), whereas the other three cells showing both L- and T-type ICa were in the G1 phase. From culture days 1 to 12, during which time cultured cells were proliferating, cells in the G1, S, or M phase increased, and the cells showing T-type ICa increased (14% at day 1 and 40% at day 2 in Fig 3⇓). During this period, all cells in the G0 phase showed only L-type ICa (n=56). After >12 days, the cultured cells generally reached confluence, and all cells showed only L-type ICa (n=48). The immunocytochemical analysis of the cell cycle, which was performed in 32 of 48 cells, showed that all cells were in the G0 phase.
Characteristics of L-Type ICa in the G0, G1, and M Phases
The characteristics of L-type ICa were compared among the different phases of the cell cycle. We first examined the voltage-dependent properties of L-type ICa. Among the G0, G1, and M phases, there were no significant differences in the current-voltage relationship (Fig 4a⇓), the steady state inactivation (Fig 4b⇓), or the steady state activation (Fig 4c⇓) of L-type ICa.
Second, we examined the effect of Bay K 8644, a dihydropyridine Ca2+ channel agonist, on L-type ICa. Bay K 8644 concentration-dependently increased L-type ICa, and the maximum effects were achieved at a concentration of >1 μmol/L. The concentration-response relationships of the Bay K 8644-induced augmentation of L-type ICa were similar among the cell-cycle phases (Fig 4d⇑). When L-type ICa was maximally augmented by Bay K 8644 (1 to 10 μmol/L), the current density of L-type ICa was significantly greater in the G1 phase (46.1±11.7 μA/cm2) than in the G0 phase (14.9±3.5 μA/cm2) or the M phase (13.5±2.3 μA/cm2) (P<.05). We also evaluated the inhibitory effect of nifedipine, a dihydropyridine Ca2+-channel antagonist, on L-type ICa. No significant difference was observed in the concentration-response relationship of the nifedipine-induced inhibition of L-type ICa among the different phases of the cell cycle (data not shown).
Third, we examined the effect of second messengers on L-type ICa. PDBu, a protein kinase C-activating phorbol ester, increased L-type ICa in a concentration-dependent manner. No significant difference was found in the concentration-response relationship of the PDBu-induced augmentation between the phases of the cell cycle (Fig 4e⇑). Dibutyryl cGMP, a membrane-permeable analogue of cGMP, inhibited L-type ICa in a concentration-dependent manner. No significant difference was observed in the concentration-response relationship of the cGMP-induced inhibition between the phases of the cell cycle (Fig 4f⇑). In this preparation, dibutyryl cAMP (10 to 1000 μmol/L), a membrane-permeable analogue of cAMP, had no effects on L-type ICa in any phase of the cell cycle (data not shown).
The present study demonstrated for the first time the cell cycle-dependent expression of L- and T-type ICa using vascular smooth muscle cells in primary culture. The characteristics of L-type ICa did not differ between the phases of the cell cycle. The selective expression of T-type ICa in the G1 and S phases, but not in the G0 phase, is also consistent with the results of the previous studies demonstrating the increased expression of T-type ICa in the proliferating cells.1 2 3 4 5 6 7 8 9 The cell cycle-dependent expression of T- and L-type ICa can also explain the finding that the expression of the two types of ICa changes with the duration of culture in this preparation.10 11 All cells within 6 hours of culture, which were considered to be freshly dispersed cells, were in the G0 phase and showed only L-type ICa. Therefore, smooth muscle cells in the rat aorta in vivo may be in the G0 phase and have only L-type Ca2+ channels. Moreover, all cells in the G0 phase showed only L-type ICa regardless of the difference in the duration of culture, thus indicating that the expression of L- and T-type ICa does not depend on the duration or the state of cell culture, but instead depends on the cell cycle.
The difference in the density of L-type ICa could be the result of either the change in the open probability or the change in the number of the channels. We cannot clarify which mechanism might be responsible, but it appears unlikely that the difference in the current density resulted from the change in the open probability. First, we examined the density of L-type ICa after the maximum augmentation of the open probability of L-type Ca2+ channels by Bay K 8644.21 Even after the maximum augmentation of open probability, the density of L-type ICa was greater in the G1 phase than in the G0 or M phase. Second, we examined the density of L-type ICa after modification of the open probability by protein kinases. It has been shown that the open probability of L-type Ca2+ channels may be modified by protein kinases.14 22 Protein kinase C14 increases and protein kinase G22 decreases the open probability of L-type Ca2+ channels. It also has been shown that the activity of protein kinases changes with the cell-cycle progression.23 However, the effects of PDBu (an activator of protein kinase C) or dibutyryl cGMP on L-type ICa did not differ among the phases of the cell cycle. It is known that cAMP potentiates L-type ICa in cardiac myocytes. In smooth muscle cells, however, the findings on the effect of cAMP on L-type ICa are not consistent. Potentiation,24 inhibition,25 dual effects (potentiation and inhibition),22 and the absence of any effect26 have been reported. The tissue diversity and the difference in experimental conditions may account for the reported differences in the modulation of L-type ICa by cAMP in smooth muscle cells. In our preparation, dibutyryl cAMP (up to 1 mmol/L) had no significant effect on L-type ICa at any phase of the cell cycle. These findings suggest that the modification of the open probability of L-type ICa by protein kinases cannot explain the cell cycle-dependent difference in the density of L-type ICa. Therefore, we consider the possibility that the cell cycle-dependent changes in the density of L-type ICa may be due to the changes in the channel number, as previously suggested in the cell cycle-dependent changes in other ion channels.15 17 18
The major implications of the present study are as follows: First, the cell cycle-dependent expression of L-type Ca2+ channels may influence the smooth muscle functions related to Ca2+ influx, such as contraction, because the L-type Ca2+ channels play a major role in the Ca2+ influx in smooth muscle cells. Second, the predominant expression of T-type Ca2+ channels in the G1 and S phases but not in the G0 phase suggests that T-type Ca2+ channels may play a role in the cellular proliferation, although the precise role of T-type Ca2+ channels in smooth muscle cells remains unknown at present. Further studies are needed to clarify the precise role of T-type Ca2+ channels. Third, the cell cycle-dependent expression of Ca2+ channels must be taken into consideration when the cellular characteristics relating Ca2+ channels in the cultured or proliferating cells are investigated.
In summary, this is the first study that has directly demonstrated the cell cycle-dependent expression of Ca2+ channels. Our findings are potentially important in the understanding of the pathophysiology of various disease states, such as atherosclerosis, restenosis after angioplasty, and vascular spasm, because the proliferation of vascular smooth muscle cells plays an important role in those diseases and because the Ca2+ channels serve diverse biological functions.
Selected Abbreviations and Acronyms
|I-V||=||current (I)-voltage (V)|
|PCNA||=||proliferating cell nuclear antigen|
|V0.5||=||half-maximal activation (inactivation)|
This study was supported in part by a grant from the Japan Society for the Promotion of Science for Japanese Junior Scientists; by a grant from the Kaibara Morikazu Medical Science Promotion Foundation; by a grant from the Kanae Foundation of Research for New Medicine; by Kimura Memorial Heart Foundation Research Grant for 1995; and by grants-in-aid for Developmental Scientific Research (No. 06557045), for General Scientific Research (Nos. 07407022 and 07833008), for Scientific Research on Priority Areas (Nos. 06274221 and 06262219), and for Creative Basic Research “Studies of Intracellular Signaling Network” from the Ministry of Education, Science and Culture, Japan. We thank Brian Quinn for reading the manuscript.
- Received December 15, 1995.
- Accepted April 8, 1996.
Kawano S, DeHaan RL. Low-threshold current is major calcium current in chick ventricle cells. Am J Physiol. 1989;256:H1505-H1508.
Furukawa T, Ito H, Nitta J, Tsujino M, Adachi S, Hiroe M, Marumo F, Sawanobori T, Hiraoka M. Endothelin-1 enhances calcium entry through T-type calcium channels in cultured neonatal rat ventricular myocytes. Circ Res. 1992;71:1242-1253.
Beam KG, Knudson CM. Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle. J Gen Physiol. 1988;91:799-815.
Nuss BH, Houser SR. T-type Ca2+ current is expressed in hypertrophied adult feline left ventricular myocytes. Circ Res. 1993;73:777-782.
Xu X, Best PM. Increase in T-type calcium current in atrial myocytes from adult rats with hormone-secreting tumors. Proc Natl Acad Sci U S A. 1990;87:4655-4659.
Kuga T, Sadoshima J, Tomoike H, Kanaide H, Akaike N, Nakamura M. Actions of Ca2+ antagonists on two types of Ca2+ channels in rat aorta smooth muscle cells in primary culture. Circ Res. 1990;67:469-480.
Hirakawa Y, Kuga T, Kobayashi S, Kanaide H, Takeshita A. Dual regulation of L-type Ca2+ channels by serotonin 2 receptor stimulation in vascular smooth muscle cells. Am J Physiol. 1995;268:H544-H549.
Block ML, Moody WJ. A voltage-dependent chloride current linked to the cell cycle in Ascidian embryos. Science. 1990;247:1090-1092.
Bubien JK, Kirk KL, Rado TA, Frizzell RA. Cell cycle dependence of chloride permeability in normal and cystic fibrosis lymphocytes. Science. 1990;248:1416-1419.
Takahashi A, Yamaguchi H, Miyamoto H. Change in K+ current of HeLa cells with progression of the cell cycle studied by patch-clamp technique. Am J Physiol. 1993;265:C328-C336.
Kobayashi S, Nishimura J, Kanaide H. Cytosolic Ca2+ transients are not required for platelet-derived growth factor to induce cell cycle progression of vascular smooth muscle cells in primary culture. J Biol Chem. 1994;269:9011-9018.
Ishikawa T, Hume JR, Keef KD. Regulation of Ca2+ channels by cAMP and cGMP in vascular smooth muscle cells. Circ Res. 1993;73:1128-1137.
Huang M, Chida K, Kamata N, Nose K, Kato M, Homma Y, Takenawa T, Kuroki T. Enhancement of inositol-phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts. J Biol Chem. 1988;263:17975-17980.
Ohya Y, Kitamura K, Kuriyama H. Modulation of ionic currents in smooth muscle balls of the rabbit intestine by intracellularly perfused ATP and cyclic AMP. Pflugers Arch. 1987;408:465-473.